U.S. patent number 8,956,608 [Application Number 13/910,834] was granted by the patent office on 2015-02-17 for method for treating sarcopenia using antagonist antibodies against gdf-8.
This patent grant is currently assigned to Wyeth LLC. The grantee listed for this patent is Wyeth LLC. Invention is credited to David S. Howland, Riyez Karim, Pamela Kelley, Seung Poon Kwak, Menelas N. Pangalos, Xiang-Yang Tan, Lioudmila Tchistiakova, Frank S. Walsh, Margaret M. Zaleska.
United States Patent |
8,956,608 |
Walsh , et al. |
February 17, 2015 |
Method for treating sarcopenia using antagonist antibodies against
GDF-8
Abstract
The disclosure provides novel molecules related to growth and
differentiation factor-8 (GDF-8), in particular mouse and humanized
antibodies, and antibody fragments, including those that inhibit
GDF-8 activity and signaling in vitro and/or in vivo. The
disclosure also provides methods for diagnosing, treating,
ameliorating, preventing, prognosing, or monitoring degenerative
orders of muscle, bone, and insulin metabolism, etc., in particular
amyotrophic lateral sclerosis (ALS). In addition, the disclosure
provides pharmaceutical compositions for the treatment of such
disorders by using the antibodies, polypeptides, polynucleotides,
and vectors of the invention.
Inventors: |
Walsh; Frank S. (Bryn Mawr,
PA), Zaleska; Margaret M. (Narberth, PA), Howland; David
S. (Yardley, PA), Tchistiakova; Lioudmila (Andover,
MA), Karim; Riyez (North Andover, MA), Kelley; Pamela
(Reading, MA), Tan; Xiang-Yang (Reading, MA), Kwak; Seung
Poon (Ewing, NJ), Pangalos; Menelas N. (Pennington,
NJ) |
Applicant: |
Name |
City |
State |
Country |
Type |
Wyeth LLC |
Madison |
NJ |
US |
|
|
Assignee: |
Wyeth LLC (Madison,
NJ)
|
Family
ID: |
37606840 |
Appl.
No.: |
13/910,834 |
Filed: |
June 5, 2013 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20140086920 A1 |
Mar 27, 2014 |
|
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
13693995 |
Dec 4, 2012 |
8496934 |
|
|
|
13030978 |
Jan 8, 2013 |
8349327 |
|
|
|
12508618 |
Mar 22, 2011 |
7910107 |
|
|
|
11503062 |
Feb 15, 2011 |
7888486 |
|
|
|
60709704 |
Aug 19, 2005 |
|
|
|
|
Current U.S.
Class: |
424/133.1;
424/158.1 |
Current CPC
Class: |
A61P
3/00 (20180101); C07K 16/22 (20130101); A61P
35/00 (20180101); C07K 16/24 (20130101); A61P
5/18 (20180101); A61P 25/28 (20180101); A61P
19/00 (20180101); A61P 19/08 (20180101); A61P
15/08 (20180101); A61P 21/04 (20180101); A61P
5/50 (20180101); A61P 11/00 (20180101); A61P
3/10 (20180101); A61P 3/06 (20180101); A61P
1/00 (20180101); A61P 3/02 (20180101); A61P
19/02 (20180101); G01N 33/6896 (20130101); A61P
21/00 (20180101); G01N 33/74 (20130101); A61P
3/04 (20180101); A61P 25/00 (20180101); A61P
43/00 (20180101); A61P 19/10 (20180101); C07K
2317/92 (20130101); C07K 2317/34 (20130101); C07K
2317/565 (20130101); C07K 2317/24 (20130101); C07K
2317/76 (20130101); G01N 2800/2835 (20130101); G01N
2800/50 (20130101); G01N 2333/475 (20130101); G01N
2800/52 (20130101); A61K 2039/505 (20130101) |
Current International
Class: |
A61K
39/395 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
1 411 064 |
|
Apr 2004 |
|
EP |
|
1 915 397 |
|
Apr 2008 |
|
EP |
|
WO 94/21681 |
|
Sep 1994 |
|
WO |
|
WO 96/33735 |
|
Oct 1996 |
|
WO |
|
WO 96/34015 |
|
Oct 1996 |
|
WO |
|
WO 96/34096 |
|
Oct 1996 |
|
WO |
|
WO 97/10847 |
|
Mar 1997 |
|
WO |
|
WO 98/33887 |
|
Aug 1998 |
|
WO |
|
WO 99/02667 |
|
Jan 1999 |
|
WO |
|
WO 99/06559 |
|
Feb 1999 |
|
WO |
|
WO 99/24618 |
|
May 1999 |
|
WO |
|
WO 99/40181 |
|
Aug 1999 |
|
WO |
|
99/56768 |
|
Nov 1999 |
|
WO |
|
WO 00/43781 |
|
Jul 2000 |
|
WO |
|
WO 01/05820 |
|
Jan 2001 |
|
WO |
|
WO 03/072714 |
|
Sep 2003 |
|
WO |
|
WO 03/072715 |
|
Sep 2003 |
|
WO |
|
WO 2004/037861 |
|
May 2004 |
|
WO |
|
WO 2004/039948 |
|
May 2004 |
|
WO |
|
WO 2004/108157 |
|
Dec 2004 |
|
WO |
|
WO 2006/102574 |
|
Sep 2006 |
|
WO |
|
WO 2006/107611 |
|
Oct 2006 |
|
WO |
|
Other References
Al-Lazikani et al., "Standard Conformations for the Canonical
Structures of Immunoglobulins", J. Mol. Bioi., 273:927-48 (1997).
cited by applicant .
Akpan et al., "The effects of a soluble activin type IIB receptor
sensitivity", Int. J. Obes. (Lond)., 33(11)1265-73 (2009). cited by
applicant .
Alfarano et al., "The Biomolecular Interaction Network Database and
related tools 2005 update", Nuc. Acids Res. Database, Issue
33:D418-24 (2005). cited by applicant .
Attwood, "Genomics: The Babel of Bioinformatics", Science,
290:471-473 (2000). cited by applicant .
Azzouz et al., "VEGF delivery with retrogradely transported
lentivector prolongs survival in a mouse ALS model", Nature,
429:413-17 (2004). cited by applicant .
Bellinge et al., "Myostatin and Its Implications on animal
breeding: a review", Animal Genetics, 36:1-6 (2005). cited by
applicant .
Bending, "Humanization of Rodent Monoclonal Antibodies by CDR
Grafting", Methods: A Companion to Methods in Enzymology,
8:83-93(1895). cited by applicant .
Bogdanovich et al., "Functional improvement of dystrophic muscle by
myostatin blockade", Nature, 420:418-21 (2002). cited by applicant
.
Brown et al., "Physicochemical Activation of Recombinant Latent
Transforming Growth Factor-beta's 1, 2, and 3", Growth Factors,
3:35-43 (1999). cited by applicant .
Bruijn et al., "Unraveling the Mechanisms Involved in Motor Neuron
Degeneration in ALS", Annu. Rev. Neurosci., 27:723-49 (2004). cited
by applicant .
Casas et al., "Association of the Muscle Hypertrophy Locus with
Carcass Traits in Beef Cattle", J. Anim. Sci., 76:468-473 (1998).
cited by applicant .
Casset et al., "A peptide mimetic of an anti-CD4 monoclonal
antibody by rational design", Biochem. Biophys. Res. Commun.,
307(1):198-205 (2003). cited by applicant .
Chao, "Retrograde Transport Redux", Neuron, 39:1-2 (2003). cited by
applicant .
Chen et al., "Selection and analysis of an optimized anti-VEGF
antibody: crystal structure of an affinity-matured Fab in complex
with antigen", J. Mol. Biol., 293(4):865-81 (1999). cited by
applicant .
Clement et al., "Wild-Type Nonneuronal Cells Extend Survival of
SOD1 Mutant Motor Neurons in ALS Mice", Science, 302:113-17 (2003).
cited by applicant .
Davis et al., "Crystal structure of prostate-specific membrane
antigen, a tumor marker and peptidase", Proc. Natl. Acad. Sci.
U.S.A., 102:5981-86 (2005). cited by applicant .
Dennler et al., "Direct binding of Smad3 and Smad4 to critical
TGFI3-inducible elements in the promoter of human plasminogen
activator inhibitor-type 1 gene", EMBO J., 17:3091-3100 (1998).
cited by applicant .
Dickman, S., "Gene Mutation Provides More Meat on the Hoof".
Science, 277:1922-1923 (1997). cited by applicant .
Dobrowolny et al., "Muscle expression of a locallgf-1 isoform
protects motor neurons in an ALS mouse model", J. Cell Biol.,
168:193-99 (2005). cited by applicant .
Downer, J., (2002) "New, Better Rat Model Reveals Astrocyte Role in
ALS", UniSci (3 pgs.) Jan. 30, 2002
httQ://www.unisci.com/stories/20021/0130021.htm printed Nov. 29,
2007. cited by applicant .
Dunlop et al., "Impaired Spinal Cord Glutamate Transport Capacity
and Reduced Sensitivity to Riluzole in a Transgenic Superoxide
Dismutase Mutant Rat Model of Amyotrophic Lateral Sclerosis", J.
Neurosci., 23:1688:96 (2003). cited by applicant .
Dupuis et al., "Evidence for defective energy homeostasis in
amyotrophic lateral sclerosis: Benefit of a high-energy diet in a
transgenic mouse model", Proc. Natl. Acad. Sci. U.S.A.,
101:11159-64 (2004). cited by applicant .
Fischer et al., "Amyotrophic lateral sclerosis in a distal
axonapthy: evidence in mice and man", Exp. Neurol., 185:232-40
(2004). cited by applicant .
Frev et al., "Early and Selective Loss of Neuromuscular Synapse
Subtypes with Low Sprouting Competence in Motoneuron Diseases", J.
Neurosci., 20:2534-42 (2000). cited by applicant .
Gamer et al., "A Novel BMP Expressed in Developing Mouse Limb,
Spinal Cord, and Tail Bud is a Potent Mesoderm Inducer in Xenopus
Embryos", Dev. Bioi., 208:222-32 (1999). cited by applicant .
Gardlik et al., "Vectors and delivery systems in gene therapy",
Med. Sci. Monit., 11:RA110-21 (2005). cited by applicant .
Gonzalez-Cadavid et al., "Organization of the human myostatin gene
and expression in healthy men and HIV-infected men with muscle
wasting", Proc. Natl. Acad. Sci. U.S.A., 95:14938-43 (1998). cited
by applicant .
Grobet et al., "A Deletion in the Bovine Myostatin Gene Causes the
Double-Muscled Phenotype in Cattle", Nature Genetics, 17:71-74
(1997). cited by applicant .
Guo et al., "Increased expression of the glial glutamate
transporter EAA T2 modulates excitotoxicity and delays the onset
but not the outcome of ALS in mice", Hum. Mol. Genet., 12:2519-32
(2003). cited by applicant .
Guo et al., "Myostatin inhibition in muscle, but not adipose
tissue, decreases fat mass and improves insulin sensitivity", PLoS
One, 4(3):e4937 (2009). cited by applicant .
Halipin and Harbury, "DNA Display II. Genetic Manipulation of
Combinatorial Chemistry Libraries for Small-Molecule Evolution",
PLoS Biology, 2:1 022-30 (2004). cited by applicant .
Hamrick et al., "Femoral Morphology and Cross-sectional Geometry of
Adult Myostatin-Deficient Mice", Bone, 27:343-49 (2000). cited by
applicant .
Hamrick et al., "Bone architecture and disc degeneration in the
lumbar spine of mice lacking GDF-8 (myostatin)", J. Orthop. Res.,
21:6):1025-32 (2003). cited by applicant .
Hamrick et al., "Bone mineral content and density in the humerus of
adult myostatin-deficient mice", Calcif. Tissue Int., 71(1):63-8
(2002). cited by applicant .
Hamrick et al., "Recombinant myostatin (GDF-8) propeptide enhances
the repair and regeneration of both muscle and bone in a model of
deep penetrant musculoskeletal injury", J. Trauma, 69(3):579-83
(2010). cited by applicant .
Hamrick M.W., "Increased bone mineral density in the femoral of
GDF8 knockout mice", Anat. Rec. A Discov. Mol. Cell. Evol. Biol.,
272(1):388-91 (2003). cited by applicant .
Holzbaur et al., "Myostatin inhibition slows muscle atrophy in
rodent models of amyotrophic lateral sclerosis", Neurobiol. Dis.,
23:697-707 (2006). cited by applicant .
Holzbaur, "Motor neurons rely on motor proteins", Trends Cell
Biol., 14:233-40 (2004). cited by applicant .
Hoodless and Wrana, "Mechanism and Function of Signaling by the
TGF.beta. Superfamily", Curr. Top. Microbial. Immunol., 228:235-72
(1998). cited by applicant .
Howland et al., "Focal loss of the glutamate transporter EAA T2 in
a transgenic rat model of SOD1 mutant-mediated amyotrophic lateral
sclerosis (ALS)", Proc. Natl. Acad. Sci. U.S.A., 99:1604-09 (2002).
cited by applicant .
Kambadur et al., "Mutations in myostatin (GDF8) in Double-Muscled
Belgian Blue and Piedmontese Cattle", Genome Res., 7:910-15 (1997).
cited by applicant .
Karp, "An ontology for biological function based on molecular
interactions", Bioinformatics Ontology, 16:269-85 (2000). cited by
applicant .
Kaspar et al., "Retrograde Viral Delivery of IGF-1 Prolongs
Survival in a Mouse ALS Model", Science, 301:839-42 (2003). cited
by applicant .
Kellum et al., "Myostatin (GDF-8) deficiency increases fracture
callus size, Sox-5 expression, and callus bone volume", Bone,
44(1):17-23 (2009). cited by applicant .
Kieran et al., "Treatment with arimoclomol, a coninducer of heat
shock proteins, delays disease progress in ALS mice", Nat. Med.,
10:402-05 (2004). cited by applicant .
Kim et al., "Inhibition of Preadipocyte Differentiation by
Myostatin Treatment in 3T3-L 1 Cultures", Biochem. Biophys. Res.
Commun., 281:902-06 (2001). cited by applicant .
Lamminmaki et al., "Crystal structure of a recombinant
anti-estradiol Fab fragment in complex with 17beta-estradiol", J.
Biol. Chem., 276(39):36687-94 (2001). cited by applicant .
LaMonte et al., "Disruption of Dyneini/Dynactin Inhibits Axonal
Transport in Motor Neurons Causing Late-Onset Progressive
Degeneration", Neuron, 34:715-27 (2002). cited by applicant .
Langley et al., "Myostatin Inhibits Myoblast Differentiation by
Down-Regulating MyoD Expression", J. Biol. Chem., 277:49831-40
(2002). cited by applicant .
Lechtzin et al., "Amyotrophic lateral sclerosis: evaluation and
treatment of respiratory impairment", Amyotroph. Lateral Seler.
Other Motor Neuron Disord., 3:5-13 (2002). cited by applicant .
Lee and McPherron, "Myostatin and the control of skeletal muscle
mass", Curr. Opin. Genet. Dev., 9:604-07 (1999). cited by applicant
.
Ligon et al., "Mutant superoxide dismutase disrupts cytoplasmic
dynein in motor neurons," Neuro Report, 16:533-36 (2005). cited by
applicant .
MacCallum et al., "Antibody-antigen interactions: contact analysis
and binding site topography", J. Mol. Biol., 262(5):732-45 (1996).
cited by applicant .
McCroskery et al., "Improved muscle healing through enhanced
regeneration and reduced fibrosis in myostatin-null mice", J. Cell.
Sci., 118:3531-41 (2005). cited by applicant .
McCroskery et al., "Myostatin negatively regulates satellite cell
activation and self-renewal", J. Cell Biol., 162:1135-47 (2003).
cited by applicant .
McKnight, Steven L., "Gatekeepers of Organ Growth", Proc. Natl.
Acad. Sci. U.S.A., 94:12249-12250 (1997). cited by applicant .
McPherron and Lee, "Double muscling in cattle due to mutations in
the myostatin gene", Proc. Natl. Acad. Sci. U.S.A., 94:12457-61
(1997). cited by applicant .
McPherron and Lee, "Supression of body fat accumulation in
myostatin-deficient mice", J. Clin. Invest., 109(5):595-601 (2002).
cited by applicant .
McPherron et al., "Regulation of skeletal muscle mas in mice by a
new TGF-13 superfamily member", Nature, 387:88-90 (1997). cited by
applicant .
McPherron et al., "Soluble activin receptor type IIB treatment does
not cause fat loss in mice with diet-induced obesity", Diabetes
Obes. Metab., Mar. 2012;14(3):279-82. Epub Nov. 21, 2011. cited by
applicant .
Mennissier, F., "Present State of Knowledge About the Genetic
Determination of Muscular Hypertrophy or the Double Muscled Trait
in Cattle", Muscle Hypertrophy of Genetic Origin and Its Uses to
Improve Beef Production: A Seminar in CEC Programme of Coordinated
Research on Beef Production, pp. 387-428 (1982). cited by applicant
.
Mitchell and Wall, "In vivo evaluation of changes in body
composition of transgenic mice expressing the mystatin pro domain
using dual energy X-ray absorptiometry", Growth Dev. Aging.,
70(1):25-37 (2007). cited by applicant .
Molina et al., "Improved Performances of Spot Multiple Peptide
Synthesis", Peptide Res., 9:151-55 (1996). cited by applicant .
Morgan et al., "The N-terminal end of the CH2 domain of chimeric
human IgG1 and anti-HLA-DR is necessary C1q, FcyR1 and FcyRIII
binding", Immunology, 86:319-24 (1995). cited by applicant .
Muyidermans et al., "Recognition of antigens by single-domain
antibody fragments: the superfluous luxury of paired domains",
Trends Biochem. Sci., 26:230-35 (2001). cited by applicant .
Muyldermans, "Single domain camel antibodies: current status", Rev.
Mol. Biotechnol., 74:277-302 (2001). cited by applicant .
Nakatani et al., "Follastatin-derived peptide expression in muscle
decreases adipose tissue mass and prevents hepatic steatosis", Am.
J. Physiol. Endocrinol. Metab., 300(3):E543-53 (2011). Epub Jan. 4,
2011. cited by applicant .
Padlan et al., "Structure of an antibody-antigen complex: crystal
structure of the HyHEL-10 Fab-lysozyme complex", Proc. Natl. Acad.
Sci. U.S.A., 86:5938-5942 (1989). cited by applicant .
Parkington, Bialek, Watner et al., "Mice treated with a
myostatin/GDF-8 decoy receptor, ActRIIB-Fc, exhibit a tremendous
increase in bone mass", Abstracts/Bone, 42:S17-S110, abstract No.
66 (2008). cited by applicant .
Pascalis et al., "Grafting of "abbreviated"
complementarily-determing regions containing
specificity-determining residues essential for ligand contact to
engineer a less immunogenic humanized monoclonal antibody", J.
Immunol., 169:6):3076-84 (2002). cited by applicant .
Resume et al., "Motor neurons in Cu/Zn superoxide
dismutase-deficient mice develop normally but exhibit enhanced cell
death after axonal injury", Nat. Genet., 13:43-47 (1996). cited by
applicant .
Rudikoff et al. "Single amino acid substitution altering
antigen-binding specificity", Proc. Natl. Acad. Sci. U.S.A.,
79(6):1979-83 (1982). cited by applicant .
Schier et al., "Isolation of Picomolar Affinity Anti-c-erbB-2
Single-chain Fv by Molecular Evoluation of the Complementarity
Determining Regions in the Center of the Antibody Binding Site", J.
Mol. Biol., 263:551-67 (1996). cited by applicant .
Schmitz and Hof, "Design-based sterology in neuroscience",
Neuroscience, 130:813-31 (2005). cited by applicant .
Schmitz and Hof, "Recommendations for straightforward and rigorous
methods of counting neurons based on a computer simulation
approach", J. Chem. Neuroanat., 20:93-114 (2000). cited by
applicant .
Schutz et al., "The Oral Antidiabetic Pioglitazone Protects from
Neurodegeneration and Amyotrophic Lateral Sclerosis-Like Symptoms
in Superoxide Dismutase-G93A Transgenic Mice", J. Neurosci.,
25:7805-12 (2005). cited by applicant .
Sharp, "The effect of peripheral nerve injury on disease
progression in the SOD1(G93A) mouse model of amyotrophic lateral
sclerosis", Neuroscience, 130:897-910 (2005). cited by applicant
.
Shields et al., "High Resolution Mapping of the Binding Site on
Huma IgG1 for FcyRI, FcyRII, FcyRIII, and FcRn and Design of IgG1
Variants with Improved Binding to the FcyR", J. Biol. Chem.
276:6591:604 (2001). cited by applicant .
Skolnick et al., "From genes to protein structure and function:
novel applications of computational approaches in the genomic era",
Trends in Biotechnol., 18:34-39 (2000). cited by applicant .
The Merck Manuals Online Medical Library, [online]. Whitehouse
Station, NJ: Merck Research Laboratory, 2006-2007. [retrieved on
Dec. 6, 2007]. Retrieved from the Internet: , URL: httg://www.
merck.com/mmpe/print/sec16/ch223/ch223f.html>. Motor Neuron
Disorders, see pp. 1-4. cited by applicant .
The Merck Manuals Online Medical Library, [online]. Whitehouse
Station, NJ: Merck Research Laboratories, 2006-2007. [retrieved on
Mar. 22, 2010]. Retrieved from the Internet: < URL: httg://www.
merck.com/mmpe/print/sec15/ch199/ch199b. html>. Anorexia
Nervosa: East Disorders Shock, see pp. 1-4. cited by applicant
.
Thies et al., "GDF-8 Propeptide Binds to GDF-8 and Antagonizes
Biological Activity by Inhibiting GDF-8 Receptor Binding", Growth
Factors, 18:251-59 (2001). cited by applicant .
Thomas et al., "Myostatin, a Negative Regulator of Muscle Growth,
Functions by Inhibiting Myoblast Proliferation", J. Biol. Chem.,
275:40235-43 (2000). cited by applicant .
Tobin and Celeste, "Myostatin, a negative regulator of muscle mass:
implications for muscle degenerative diseases", Curr. Opin.
Pharmacol., 5:328-32 (2005). cited by applicant .
Vajdos et al., "Comprehensive functional maps of the
antigen-binding site of an anti-ErbB2 antibody obtained with
shotgun scanning mutagenesis", J. Mol. Biol., 320(2):415-28 (2002).
cited by applicant .
van den Beucken et al., "Building Novel Binding Ligands to B7.1 and
B7.2 Based on Human Antibody Single Variable Light Chain Domains",
J. Mol. Biol., 310:591-601 (2002). cited by applicant .
Verkman, "Drug discovery in academia", Am. J. Physiol. Cell
Physiol., 286:465-74 (2004). cited by applicant .
Vukicevic et al., "Induction of nephrogenic mesenchyme by
osteogenic protein 1 (bone morphogenetic protein 7)", Proc. Natl.
Acad. Sci. U.S.A., 93:9021-9026 (1996). cited by applicant .
Wagner et al., "Loss of Myostatin Attenuates Severity of Muscular
Dystrophy in mdx Mice", Ann. Neurol., 52:832-36 (2002). cited by
applicant .
Wang et al., "Neuroprotective Effects of Glial Cell Line-Derived
Neurotrophic Factor Mediated by an Adeno-Associated Virus Vector in
a Transgenic Animal Model of Amyotrophic Lateral Sclerosis", J.
Neurosci., 22:6920-28 (2002). cited by applicant .
Ward et al., "Binding activities of a repertoire of single
immunoglobulin variable domains secreted from Escherichia coli",
Nature, 341:544-46 (1989). cited by applicant .
Whittemore et al., "Inhibition of myostatin in adult mice increases
skeletal muscle mass and strength", Biochem. Biophys. Res. Commun.,
300:965-71 (2003). cited by applicant .
Wilkes et al., "Loss-of-function mutation in mystatin reduces tumor
necrosis factor alpha production and protects liver against
obesity-induced insulin resistance", Diabetes, 58(5):1133-43
(2009). cited by applicant .
Wooley et al., "Gait analysis detects early changes in transgenic
SOD1 (G93A) mice", Muscle Nerve, 32:43-50 (2005). cited by
applicant .
Wu et al., "Humanization of a murine monoclonal antibody by
simultaneous optimization of framework and CDR residues", J. Mol.
Biol., 294(1):151-62 (1999). cited by applicant .
Zimmers et al., "Induction of Cachexia in Mice by Systemically
Administered Myostatin", Science, 296:1486-88 (2002). cited by
applicant.
|
Primary Examiner: Wen; Sharon
Attorney, Agent or Firm: DLA Piper LLP (US)
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional application of U.S. application
Ser. No. 13/693,995 filed Dec. 4, 2012, now issued as U.S. Pat. No.
8,496,934; which is a divisional of U.S. application Ser. No.
13/030,978 filed Feb. 18, 2011, now issued as U.S. Pat. No.
8,349,327; which is a divisional of U.S. application Ser. No.
12/598,618 filed Jul. 24, 2009, now issued as U.S. Pat. No.
7,910,107; which is a divisional of U.S. application Ser. No.
11/503,062 filed Aug. 14, 2006, now issued as U.S. Pat. No.
7,888,486; which claims the benefit under 35 USC .sctn.119(e) to
U.S. Application Ser. No. 60/709,704 filed Aug. 19, 2005. The
disclosure of each of the prior applications is considered part of
and is incorporated by reference in the disclosure of this
application.
Claims
What is claimed is:
1. A method of treating sarcopenia in a mammal, comprising
administering to a mammal in need of treatment for sarcopenia a
therapeutically effective amount of a GDF-8 specific antibody or
antigen binding fragment thereof, said antibody or fragment
comprising: an antibody variable heavy (VH) region comprising the
first, second and third complementarity determining regions (CDR)
from the VH region defined by the amino acid sequence of SEQ ID
NO:3 or SEQ ID NO:7; and an antibody variable light (VL) region
comprising the first, second and third complementarity determining
regions (CDR) from the VL region defined by the amino acid sequence
of SEQ ID NO:5 or SEQ ID NO:9.
2. The method of claim 1, wherein VH CDR1 comprises SEQ ID NO:10 or
SEQ ID NO:20, VH CDR2 comprises SEQ ID NO:11 or SEQ ID NO:21, VH
CDR3 comprises SEQ ID NO:12, VL CDR1 comprises SEQ ID NO:13, VL
CDR2 comprises SEQ ID NO:14, and VL CDR3 comprises SEQ ID
NO:15.
3. The method of claim 1, wherein the VH region of said antibody or
fragment comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID
NO:7.
4. The method of claim 1, wherein the VL region of said antibody or
fragment comprises the amino acid sequence of SEQ ID NO:5 or SEQ ID
NO:9.
5. The method of claim 1, wherein the VH region of said antibody or
fragment comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID
NO:7 and the VL region of said antibody or fragment comprises the
amino acid sequence of SEQ ID NO:5 or SEQ ID NO:9.
6. The method of claim 1, wherein the VH region of said antibody or
fragment comprises the amino acid sequence of SEQ ID NO:7 and the
VL region of said antibody or fragment comprises the amino acid
sequence of SEQ ID NO:9.
7. The method of claim 1, wherein said antibody or fragment is
partially or fully humanized.
8. The method of claim 1, wherein said antibody binds to GDF-8 with
an affinity of about 10 nM or higher affinity.
9. The method fragment of claim 1, wherein said fragment is
selected from the group consisting of an Fd fragment, an Fab
fragment, an F(ab')2 fragment, an scFv fragment, and an Fv
fragment.
10. The method of claim 1, wherein said antibody or fragment
further comprises an antibody constant heavy region from a human
immunoglobulin subtype selected from the group consisting IgG1,
IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE and IgM.
11. The method or fragment of claim 10, wherein the antibody
constant heavy region is from human IgG1.
12. The method of claim 10, wherein the constant heavy region of
said antibody or fragment is modified to alter a constant region
effector function.
13. The method of claim 11, wherein said constant heavy region
comprises the amino acid sequence of SEQ ID NO:19.
14. The method of claim 13, wherein the amino acid sequence of SEQ
ID NO:19 is modified at one or more residues altering a constant
heavy region effector function.
15. The method of claim 13, wherein the amino acid sequence of SEQ
ID NO:19 is modified at least at residue 117 or 120 thereof to
alter an Fc region effector function.
16. The method of claim 1, wherein said antibody or fragment
further comprises a human antibody kappa or lambda constant light
region.
17. The method of claim 16, wherein said constant light region
comprises the amino acid sequence of SEQ ID NO:17.
18. A method of treating sarcopenia in a mammal, comprising
administering to a mammal in need of treatment for sarcopenia a
therapeutically effective amount of a GDF-8 specific antibody
comprising: two antibody heavy chains, each comprising a variable
heavy region defined by the amino acid sequence of SEQ ID NO:7 and
the constant heavy region from human IgG1; and two antibody light
chains, each comprising a variable light region defined by the
amino acid sequence of SEQ ID NO:9 and a constant light region
defined by the amino acid sequence of SEQ ID NO:17.
19. The method of claim 18, wherein said constant heavy region
comprises the amino acid sequence of SEQ ID NO:19 modified at least
at residue 117 or 120 thereof to alter an Fc region effector
function.
Description
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been
submitted via EFS-Web and is hereby incorporated by reference in
its entirety. Said ASCII copy, created on Jun. 4, 2013, is named
PFIZER1250-5_ST25.txt and is 23 KB in size.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The technical field of the invention relates to growth and
differentiation factor-8 (GDF-8) antagonists, in particular,
antibodies against GDF-8, e.g., mouse, human and humanized
antibodies and their fragments, particularly those that inhibit
GDF-8 activity in vitro and/or in vivo. The field further relates
to treating, ameliorating, preventing, prognosing, or monitoring
GDF-8-associated disorders, e.g., muscle disorders, neuromuscular
disorders, bone degenerative disorders, metabolic or induced bone
disorders, adipose disorders, glucose metabolism disorders or
insulin-related disorders, particularly amyotrophic lateral
sclerosis ("ALS").
2. Related Background Art
Growth and differentiation factor-8 (GDF-8), also known as
myostatin, is a secreted protein and member of the transforming
growth factor-beta (TGF-.beta.) superfamily of structurally related
growth factors. Members of this superfamily possess
growth-regulatory and morphogenetic properties (Kingsley et al.
(1994) Genes Dev. 8:133-46; Hoodless et al. (1998) Curr. Topics
Microbiol. Immunol. 228:235-72). Human GDF-8 is synthesized as a
375 amino acid precursor protein that forms a homodimer complex.
During processing, the amino-terminal propeptide, known as the
"latency-associated peptide" (LAP), is cleaved and may remain
noncovalently bound to the homodimer, forming an inactive complex
designated the "small latent complex" (Miyazono et al. (1988) J.
Biol. Chem. 263:6407-15; Wakefield et al. (1988; J. Biol. Chem.
263:7646-54; Brown et al. (1999) Growth Factors 3:35-43; Thies et
al. (2001) Growth Factors 18:251-59; Gentry et al. (1990)
Biochemistry 29:6851-57; Derynck et al. (1995) Nature 316:701-05;
Massague (1990) Ann. Rev. Cell Biol. 12:597-641). Proteins such as
follistatin and its relatives also bind mature GDF-8 homodimers and
inhibit GDF-8 biological activity (Gamer et al. (1999) Dev. Biol.
208:222-32).
An alignment of the deduced GDF-8 amino acid sequence from various
species demonstrates that GDF-8 is highly conserved (McPherron et
al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:12457-61). The
sequences of human, mouse, rat, porcine, and chicken GDF-8 are 100%
identical in the C-terminal region, while baboon, bovine, and ovine
GDF-8 differ by a mere 3 amino acids at the C-terminus. The high
degree of GDF-8 conservation across species suggests that GDF-8 has
an essential physiological function.
GDF-8 has been shown to play a major role in the regulation of
muscle development and homeostasis by inhibiting both proliferation
and differentiation of myoblasts and satellite cells (Lee and
McPherron (1999) Curr. Opin. Genet. Dev. 9:604-07; McCroskery et
al. (2003) J. Cell. Biol. 162:1135-47). It is expressed early in
developing skeletal muscle, and continues to be expressed in adult
skeletal muscle, preferentially in fast twitch types. Additionally,
GDF-8 overexpressed in adult mice results in significant muscle
loss (Zimmers et al. (2002) Science 296:1486-88). Also, natural
mutations that render the GDF-8 gene inactive have been shown to
cause both hypertrophy and hyperplasia in both animals and humans
(Lee and McPherron (1997) supra). For example, GDF-8 knockout
transgenic mice are characterized by a marked hypertrophy and
hyperplasia of the skeletal muscle and altered cortical bone
structure (McPherron et al. (1997) Nature 387:83-90; Hamrick et al.
(2000) Bone 27:343-49). Similar increases in skeletal muscle mass
are evident in natural GDF-8 mutations in cattle (Ashmore et al.
(1974) Growth 38:501-07; Swatland et al. (1994) J. Anim. Sci.
38:752-57; McPherron et al., supra; Kambadur et al. (1997) Genome
Res. 7:910-15). In addition, various studies indicate that
increased GDF-8 expression is associated with HIV-induced muscle
wasting (Gonzalez-Cadavid et al. (1998) Proc. Natl. Acad. Sci.
U.S.A. 95:14938-43). GDF-8 has also been implicated in the
production of muscle-specific enzymes (e.g., creatine kinase) and
myoblast proliferation (WO 00/43781).
In addition to its growth-regulatory and morphogenetic properties,
GDF-8 is believed to participate in numerous other physiological
processes, including glucose homeostasis during type 2 diabetes
development, impaired glucose tolerance, metabolic syndromes (i.e.,
a syndrome such as, e.g., syndrome X, involving the simultaneous
occurrence of a group of health conditions, which may include
insulin resistance, abdominal obesity, dyslipidemia, hypertension,
chronic inflammation, a prothrombotic state, etc., that places a
person at high risk for type 2 diabetes and/or heart disease),
insulin resistance (e.g., resistance induced by trauma such as
burns or nitrogen imbalance), and adipose tissue disorders (e.g.,
obesity, dyslipidemia, nonalcoholic fatty liver disease, etc.) (Kim
et al. (2000) Biochem. Biophys. Res. Comm. 281:902-06).
A number of human and animal disorders are associated with
functionally impaired muscle tissue, e.g., amyotrophic lateral
sclerosis ("ALS"), muscular dystrophy ("MD"; including Duchenne's
muscular dystrophy), muscle atrophy, organ atrophy, frailty,
congestive obstructive pulmonary disease (COPD), sarcopenia,
cachexia, and muscle wasting syndromes caused by other diseases and
conditions. Currently, few reliable or effective therapies exist to
treat these disorders. The pathology of these diseases indicates a
potential role for GDF-8 signaling as a target in the treatment of
these diseases.
ALS is a late onset and fatal neurodegenerative disease
characterized by degeneration of the central nervous system and
muscle atrophy. ALS typically initiates with abnormalities in gait
and loss of dexterity, and then progresses to paralysis of limbs
and diaphragm. While most cases of ALS are sporadic and are of
unknown etiology, 5-10% of cases have been shown to result from
dominant familial (FALS) inheritance. Approximately 10-20% of FALS
cases are attributed to mutations in the Cu/Zn superoxide dismutase
(SOD1) gene (reviewed in Bruijn et al. (2004) Ann. Rev. Neurosci.
27:723-49). SOD1 is a heterodimeric metallo-protein that catalyzes
the reaction of superoxide into hydrogen peroxide and diatomic
oxygen, and as loss of SOD1 does not result in motor neuron disease
(Reaume et al. (1996) Nat. Genet. 13:43-47), it is believed to
induce disease by toxic gain of function (reviewed in Bruijn et
al., supra). The specific mechanisms of SOD 1-induced neuronal cell
death are unclear, and may involve alterations in axonal transport,
cellular responses to misfolded protein, mitochondrial dysfunction,
and excitotoxicity (Bruijn et al., supra).
The degeneration of motor neurons observed in ALS may occur via
multiple mechanisms, including uptake or transport disruption of
trophic factors by motor neurons (reviewed in Holzbaur (2004)
Trends Cell Biol. 14:233-40). Thus, ALS might be treated by
therapies that rejuvenate a degenerating neuron by providing an
optimal survival environment. A nerve's environment includes
normeuronal cells such as glia and the muscle cells innervated by
the motor neuron. This environment provides trophic and growth
factors that are endocytosed by the neuron and transported via
retrograde axonal transport to the cell body (Chao (2003) Neuron
39:1-2; Holzbaur, supra).
FALS has been modeled in both mouse and rat by the overexpression
of mutant SOD1 (Howland et al. (2002) Proc. Natl. Acad. Sci. U.S.A.
99:1604-09). Transgenic mice overexpressing the G93A form of mutant
SOD1 display muscle weakness and atrophy by 90 to 100 days of age,
and typically die near 130 days of age (Gurney et al. (1994)
Science 264:1772-75). However, the underlying SODG93 A-induced
pathology, which includes grip strength weakness and loss of
neuromuscular junctions, is significant as early as 50 days of age
(Frey et al. (2000) J. Neurosci. 20:2534-42; Fisher et al. (2004)
Exp. Neuro. 185:232-40; Ligon et al. (2005) NeuroReport 16:533-36;
Wooley et al. (2005) Muscle Nerve 32:43-50). Transgenic rats
expressing the SODG93A mutation follow a similar time course of
degeneration (Howland et al., supra). Recent work has suggested
that the development of pathology is not cell autonomous,
consistent with the hypothesis that the degeneration of motor
neurons observed in ALS occurs via multiple mechanisms, including
the disruption of uptake and transport of trophic factors by the
motor neuron (see above). Clement and coworkers have used chimeric
mice to show that wild type normeuronal cells can extend survival
of motor neurons expressing mutant SOD1 (Clement et al. (2003)
Science 302:113-17). These observations have led to the
investigation of therapies that might slow neuronal degeneration by
providing an optimal microenvironment for survival. For example,
treatment of the SODG93A mouse via direct intramuscular injection
of virally expressed growth factors (including IGF-1, GDNF and
VEGF) prolongs animal survival (Kaspar et al. (2003) Science
301:839-42; Azzouz et al. (2004) Nature 429:413-17; Wang et al.
(2002) J. Neurosci. 22:6920-28). In addition, muscle-specific
expression of a local IGF-1-specific isoform (mIGF-1) stabilizes
neuromuscular junctions, enhances motor neuron survival and delays
onset and progression of disease in the SODG93A transgenic mouse
model, indicating that direct effects on muscle can impact disease
onset and progression in transgenic SOD1 animals (Dobrowolny et al.
(2005) J. Cell Biol. 168:193-99). Links between muscle
hypermetabolism and motor neuron vulnerability have also been
reported in ALS mice, supporting the hypothesis that defects in
muscle may contribute to the disease etiology (Dupois et al. (2004)
Proc. Natl. Acad. Sci. U.S.A. 101:11159-64). Thus, enhancing muscle
growth should provide improved local support for motor neurons, and
therefore result in therapeutic benefits.
Inhibition of myostatin expression leads to both muscle hypertrophy
and hyperplasia (Lee and McPherron, supra; McPherron et al.,
supra). Myostatin negatively regulates muscle regeneration after
injury, and lack of myostatin in GDF-8 null mice results in
accelerated muscle regeneration (McCroskery et al., (2005) J. Cell.
Sci. 118:3531-41). Myostatin-neutralizing antibodies increase body
weight, skeletal muscle mass, and muscle size and strength in the
skeletal muscle of wild type mice (Whittemore et al. (2003)
Biochem. Biophys. Res. Commun. 300:965-71) and the mdx mouse, a
model for muscular dystrophy (Bogdanovich et al. (2002) Nature
420:418-21; Wagner et al. (2002) Ann. Neurol. 52:832-36).
Furthermore, myostatin antibody in these mice decreased the damage
to the diaphragm, a muscle that is also targeted during ALS
pathogenesis. It has been hypothesized that the action of growth
factors, such as HGF, on muscle may be due to inhibition of
myostatin expression (McCroskery et al. (2005), supra), thereby
helping to shift the "push and pull," or balance, between
regeneration and degeneration in a positive direction. Thus, GDF-8
inhibition presents as a potential pharmacological target for the
treatment of ALS, muscular dystrophy (MD), and other
GDF-8-associated disorders, e.g., neuromuscular disorders for which
it is desirable to increase muscle mass, strength, size, etc. With
the availability of animal models (mouse and rat) of ALS, it is
possible to test therapeutics in two different species, thus
increasing the confidence of therapeutic effects in humans in
vivo.
In addition to neuromuscular disorders in humans, there are also
growth factor-related conditions associated with a loss of bone,
such as osteoporosis and osteoarthritis, which predominantly affect
the elderly and/or postmenopausal women. In addition, metabolic
bone diseases and disorders include low bone mass due to chronic
glucocorticoid therapy, premature gonadal failure, androgen
suppression, vitamin D deficiency, secondary hyperparathyroidism,
nutritional deficiencies, and anorexia nervosa. Although many
current therapies for these conditions function by inhibiting bone
resorption, a therapy that promotes bone formation would be a
useful alternative treatment. Because GDF-8 plays a role in bone
development as well as muscular development, regulation of GDF-8 is
also an excellent pharmacological target for the treatment of
bone-degenerative disorders.
Thus, a need exists to develop compounds and methods of treatment
that contribute to an overall increase in muscle mass and/or
strength and/or bone density, etc., particularly in humans, and
particularly in those suffering from ALS and other muscle-wasting
diseases as well as bone-degenerative disorders. Generating
neutralizing antibodies and other small molecules with enhanced
affinity to GDF-8 is an excellent pharmacological approach to treat
these disorders.
SUMMARY OF THE INVENTION
The GDF-8 antagonists of the invention relate to antibodies (e.g.,
intact antibodies and antigen-binding fragments thereof), which are
referred to herein as "anti-GDF-8 antibodies" or "GDF-8
antibodies." In one embodiment, an anti-GDF-8 antibody reduces,
neutralizes, and/or antagonizes at least one GDF-8-associated
activity (i.e., "GDF-8 activity"). The present invention thus
provides methods to treat various bone, muscle, glucose and adipose
disorders associated with GDF-8 activity using these anti-GDF-8
antibodies. The present invention discloses that GDF-8 antagonists,
e.g., GDF-8 antibodies, are highly effective therapeutics when used
to treat animals suffering from ALS, and that administration of
such antibodies reduces the wasting of muscles targeted during ALS
pathology, e.g., diaphragm, gastrocnemius, etc. In addition, the
present invention discloses that these antagonists are highly
effective at increasing muscle mass and grip strength in
ALS-afflicted animals. As a result, the invention teaches that
anti-GDF-8 antibodies are effective compositions to treat
GDF-8-associated disorders, e.g., ALS, muscle wasting disorders or
other disorders that result from GDF-8 dysregulation.
In one aspect, the invention features a method of treating (e.g.,
curing, suppressing), ameliorating, or preventing (e.g., delaying
or preventing the onset, recurrence or relapse of) a
GDF-8-associated disorder in a subject. The method includes:
administering to the subject a GDF-8 antagonist, e.g., an
anti-GDF-8 antibody, in an amount sufficient to treat or prevent
the GDF-8-associated disorder. The GDF-8 antagonist, e.g., the
anti-GDF-8 antibody, can be administered to the subject alone or in
combination with other therapeutic modalities as described herein.
The GDF-8 antibody can be administered therapeutically,
prophylactically, or both. In one embodiment, the subject is a
mammal, e.g., a human suffering from a GDF-8-associated disorder,
including, e.g., bone and muscle disorders. Preferably, the subject
is a human. More preferably, the subject is a human suffering from
a GDF-8-associated disorder as described herein.
In one embodiment, the present invention provides safe and
effective therapeutic methods for diagnosing, prognosing,
monitoring, screening, treating, ameliorating, and/or preventing
GDF-8-associated disorders, e.g., muscle disorders, neuromuscular
disorders, bone-degenerative disorders, metabolic or induced bone
disorders, adipose disorders, glucose metabolism disorders, or
insulin-related disorders which include, but are not limited to,
glucose homeostasis, type 2 diabetes, impaired glucose tolerance,
metabolic syndrome (i.e., a syndrome involving the simultaneous
occurrence of a group of health conditions, which may include
insulin resistance, abdominal obesity, dyslipidemia, hypertension,
chronic inflammation, a prothrombotic state, etc., that places a
person at high risk for type 2 diabetes and/or heart disease),
insulin resistance (e.g., resistance induced by trauma such as
burns or nitrogen imbalance), adipose tissue disorders (e.g.,
obesity, dyslipidemia, nonalcoholic fatty liver disease, etc.),
HIV-induced muscle wasting, muscular dystrophy (including
Duchenne's muscular dystrophy), amyotrophic lateral sclerosis
("ALS"), muscle atrophy, organ atrophy, frailty, congestive
obstructive pulmonary disease, sarcopenia, cachexia, muscle wasting
syndromes, osteoporosis, osteoarthritis, metabolic bone diseases,
and metabolic bone disorders (including low bone mass due to
chronic glucocorticoid therapy, premature gonadal failure, androgen
suppression, vitamin D deficiency, secondary hyperparathyroidism,
nutritional deficiencies, and anorexia nervosa). In a preferred,
but not limiting, embodiment, the invention provides safe and
effective therapeutic methods for diagnosing, prognosing,
monitoring, screening, treating, ameliorating, and/or preventing a
GDF-8-associated disorder, e.g., a muscular disorder in
vertebrates, particularly mammals, and more particularly humans. In
a most preferred embodiment of the invention, the GDF-8-associated
disorder, e.g., muscle disorder, diagnosed, prognosed, monitored,
screened, treated, ameliorated, and/or prevented is ALS.
In another embodiment, this invention provides methods of
inhibiting GDF-8 function in vivo or in vitro. These methods are
useful for treating GDF-8-associated disorders, e.g., muscle and
bone degenerative disorders, particularly muscle disorders such as
ALS, and for increasing muscle mass and/or bone strength and/or
density. The methods are also useful for increasing muscle mass and
bone density in normal animals including, but not limited to,
humans. The subject methods can be used in vitro (e.g., in a
cell-free system, in culture, etc.), ex vivo, or in vivo. For
example, GDF-8 receptor-expressing cells can be cultured in vitro
in culture medium and contacted with, e.g., one or more anti-GDF-8
antibodies, e.g., as described herein. Alternatively, the method
can be performed on cells present within a subject, e.g., as part
of an in vivo (e.g., therapeutic or prophylactic) protocol.
Accordingly, in one aspect, the invention features a GDF-8
antagonist, e.g., an isolated antibody, that interacts with, e.g.,
binds to, and neutralizes and/or inhibits, GDF-8. In particular,
the GDF-8 protein bound by the GDF-8 antibody is mammalian, e.g.,
human, sheep, nonhuman primate GDF-8. In another embodiment, the
invention provides antibodies that bind GDF-8 with high affinity,
e.g., with a Kd of at least 10.sup.-7 M, preferably 10.sup.-8,
10.sup.-9, 10.sup.-10, more preferably, 10.sup.-11 M or higher. The
affinity and binding kinetics of the anti-GDF-8 antibody can be
tested using several well-known methods, e.g., biosensor technology
(Biacore, Piscataway, N.J.).
In one embodiment, the anti-GDF-8 antibody (e.g., an intact
antibody or an antibody fragment (e.g., a Fab, F(ab').sub.2, Fv or
a single chain Fv fragment)) is a monoclonal antibody. The antibody
may be a human, humanized, chimeric, or an in vitro-generated
antibody. In a preferred, but not limiting, embodiment, an
anti-GDF-8 antibody of the invention is a humanized antibody.
These anti-GDF-8 antibodies can be used to diagnose, prognose,
monitor, screen, treat, ameliorate, and/or prevent muscle, bone,
adipose and glucose metabolism-related disorders. A nonlimiting
example of an anti-GDF-8 antibody is referred to herein as "RK35,"
and includes both mouse and modified antibodies, e.g., chimeric or
humanized forms. The nucleotide and amino acid sequences for the
heavy chain variable region of mouse RK35 are set forth herein as
SEQ ID NO:2 and SEQ ID NO:3, respectively. The nucleotide and amino
acid sequences for the heavy chain variable region of humanized
RK35 are set forth herein as SEQ ID NO:6 and SEQ ID NO:7,
respectively. The nucleotide and amino acid sequences for the light
chain variable region of mouse RK35 are set forth herein as SEQ ID
NO:4 and SEQ ID NO:5, respectively. The nucleotide and amino acid
sequences for the light chain variable region of humanized RK35 are
set forth herein as SEQ ID NO:8 and SEQ ID NO:9, respectively.
In a preferred, but not limiting, embodiment of the invention, the
antibody is a mouse or humanized antibody to GDF-8. In a more
preferred embodiment of the invention, the antibody is comprised of
the VH (variable heavy) domain set forth in SEQ ID NO:3 and the VL
(variable light) domain set forth in SEQ ID NO:5. In another
preferred embodiment of the invention, the antibody is comprised of
the VH domain set forth in SEQ ID NO:7 and the VL domain set forth
in SEQ ID NO:9. Additional embodiments of the invention comprise
one or more VH or VL domains listed in Table 1.
Other embodiments of the invention comprise an H3 fragment of RK35,
i.e., the sequence set forth as SEQ ID NO:12. In yet another
embodiment, a GDF-8 antagonist comprises one, two, or three
complementarity determining regions (CDRs) from a heavy chain
variable region of an antibody disclosed herein with sequences
selected from SEQ ID NOs:10-12 and 20-22. In yet another
embodiment, an antagonist of the invention comprises one, two, or
three CDRs from a light chain variable region of an antibody
disclosed herein with sequences selected from SEQ ID NOs:13-15 and
23-25. In yet another embodiment, the antibody comprises one, two,
three, four, five, or six CDRs with sequences selected from SEQ ID
NOs:10-15 and 20-25.
The heavy and light chains of an anti-GDF-8 antibody of the
invention may be full-length (e.g., an antibody can include at
least one, and preferably two, complete heavy chains, and at least
one, and preferably two, complete light chains) or may include an
antigen-binding fragment (e.g., a Fab, F(ab').sub.2, Fv or a single
chain Fv fragment ("scFv")). In other embodiments, the antibody
heavy chain constant region is chosen from, e.g., IgG1, IgG2, IgG3,
IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly chosen from,
e.g., IgG1, IgG2, IgG3, and IgG4, more particularly, IgG1 (e.g.,
human IgG1). In another embodiment, the antibody light chain
constant region is chosen from, e.g., kappa or lambda, particularly
kappa (e.g., human kappa). In one embodiment, the constant region
is altered, e.g., mutated, to modify the properties of the antibody
(e.g., to increase or decrease one or more of: Fc receptor binding,
antibody glycosylation, the number of cysteine residues, effector
cell function, or complement function). For example, the human IgG1
constant region can be mutated at one or more residues, e.g., one
or more of residues 117 and 120 of SEQ ID NO:19. In one embodiment,
the anti-GDF-8 antibody comprises the human IgG1 constant region
shown in SEQ ID NO:19. In another embodiment, the anti-GDF-8
antibody comprises a human kappa sequence, e.g., the sequence shown
as SEQ ID NO:17.
In another embodiment, the invention provides GDF-8 antibodies as
novel antibody fragments that bind GDF-8 and retain the ability to
neutralize or reduce GDF-8 activity. In a preferred, but not
limiting, embodiment of the invention, the antibody fragment is
selected from the group consisting of a dAb fragment, a diabody, an
Fd fragment, an Fab fragment, an F(ab').sub.2 fragment, an scFV
fragment, and an Fv fragment. In a more preferred embodiment of the
invention, the antibody fragment is encoded by a polynucleotide
selected from SEQ ID NOs:2, 4, 6 or 8. In another preferred
embodiment of the invention, the antibody fragment comprises an
amino acid sequence selected from an amino acid sequence set forth
in SEQ ID NOs:10-15 and 20-25. In another preferred embodiment, the
invention provides novel antibody fragments that differ in sequence
(e.g., due to the redundancy of the genetic code) from those
sequences listed in Table 1, yet retain the ability to bind GDF-8
and neutralize or reduce GDF-8 activity.
In another embodiment, the anti-GDF-8 antibody comprises at least
one, two, three or four antigen-binding regions, e.g., variable
regions, having an amino acid sequence as listed in Table 1 (SEQ ID
NOs:3 or 7 for VH, and/or SEQ ID NOs:5 or 9 for VL), or a sequence
substantially identical thereto (e.g., a sequence at least about
85%, 90%, 95%, 99% or more identical thereto, or which differs by
no more than 1, 2, 5, 10 or 15 amino acid residues from SEQ ID
NOs:3, 5, 7 or 9). In another embodiment, the antibody includes a
VH and/or VL domain encoded by a nucleic acid having a nucleotide
sequence as listed in Table 1 (SEQ ID NOs:2 or 6 for VH, and/or SEQ
ID NOs:4 or 8 for VL), or a sequence substantially identical
thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more
identical thereto, or which differs by no more than 3, 6, 15, 30 or
45 nucleotides from SEQ ID NOs:2, 4, 6, or 8). In yet another
embodiment, the antibody comprises one, two, or three CDRs from a
heavy chain variable region having amino acid sequences as listed
in Table 1 (SEQ ID NOs:10-12 and 20-22), or a sequence
substantially homologous thereto (e.g., a sequence at least about
85%, 90%, 95%, 99% or more identical thereto, and/or having one or
more substitutions, e.g., conserved substitutions). In yet another
embodiment, the antibody comprises at least one, two, or three CDRs
from a light chain variable region having amino acid sequences as
listed in Table 1 (SEQ ID NOs:13-15 and 23-25), or a sequence
substantially homologous thereto (e.g., a sequence at least about
85%, 90%, 95%, 99% or more identical thereto, and/or having one or
more substitutions, e.g., conserved substitutions). In yet another
embodiment, the antibody comprises one, two, three, four, five or
six CDRs from heavy and light chain variable regions having amino
acid sequences as listed in Table 1 (SEQ ID NOs:10-12 and 20-22 for
VH CDRs; and SEQ ID NOs:13-15 and 23-25 for VL CDRs), or a sequence
substantially homologous thereto (e.g., a sequence at least about
85%, 90%, 95%, 99% or more identical thereto, and/or having one or
more substitutions, e.g., conserved substitutions).
In another embodiment, the anti-GDF-8 antibody comprises a human
IgG1 constant region having an amino acid sequence as set forth in
SEQ ID NO:19 or a sequence substantially homologous thereto (e.g.,
a sequence at least about 85%, 90%, 95%, 99% or more identical
thereto, or which differs by no more than 1, 2, 5, 10, 50, or 100
amino acid residues from SEQ ID NO:19). In another embodiment, the
anti-GDF-8 antibody comprises a human kappa constant chain, e.g., a
human kappa constant chain having an amino acid sequence as set
forth in SEQ ID NO:17 or a sequence substantially homologous
thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more
identical thereto, or which differs by no more than 1, 2, 5, 10,
20, or 50 amino acid residues from SEQ ID NO:17). In yet another
embodiment, the antibody comprises a human IgG1 constant region and
a human kappa constant chain as described herein.
In a preferred, but not limiting, embodiment, the invention
provides antibodies encoded by polynucleotides set forth in SEQ ID
NOs:2, 4, 6, or 8. In another preferred embodiment, the invention
provides antibodies encoded by polynucleotide sequences that
hybridize under stringent conditions to the polynucleotides set
forth in SEQ ID NOs:2, 4, 6, or 8. In another preferred embodiment,
the invention provides antibodies encoded by polynucleotides, which
differ from those sequences set forth in SEQ ID NOs:2, 4, 6, or 8,
but due to the degeneracy of the genetic code, encode an amino acid
sequence set forth in SEQ ID NOs:3, 5, 7, 9, or 10-15.
The GDF-8 antagonist, e.g., an anti-GDF-8 antibody, can be
derivatized or linked to another functional molecule, e.g., another
peptide or protein (e.g., an Fab fragment). For example, a fusion
protein or an antibody, or antigen-binding portion, can be
functionally linked (e.g., by chemical coupling, genetic fusion,
noncovalent association or otherwise) to one or more other
molecular entities, such as an antibody (e.g., a bispecific or a
multispecific antibody), toxins, radioisotopes, cytotoxic or
cytostatic agents, among others.
A further aspect of the invention provides as GDF-8 antagonists
purified and isolated nucleic acids that encode the GDF-8
antagonists, e.g., anti-GDF-8 antibodies, of the invention. In one
embodiment, the invention provides polynucleotides comprised of a
sequence encoding a VH (SEQ ID NO:3 or SEQ ID NO:7), VL (SEQ ID
NO:5 or SEQ ID NO:9), and/or CDR (SEQ ID NOs:10-15 and 20-25) as
listed in Table 1. In another embodiment, the invention provides
polynucleotides that hybridize under stringent conditions to
nucleic acids encoding a VH, VL, or CDR (SEQ ID NOs:3, 5, 7, 9,
10-15, or 20-25) as listed in Table 1. In another embodiment, the
invention provides nucleic acids that comprise SEQ ID NOs:2, 4, 6,
or 8 or fragments of SEQ ID NOs:2, 4, 6, or 8. In yet a further
embodiment, the invention provides polynucleotides that hybridize
under stringent conditions to SEQ ID NOs:2, 4, 6 or 8. Another
aspect of the invention provides host cells and vectors comprising
the polynucleotides of the invention as GDF-8 antagonists.
The antibodies of the invention possess a number of useful
properties. First, the antibodies are capable of binding mature
GDF-8 with high affinity. Second, the disclosed antibodies inhibit
GDF-8 activity in vitro and in vivo. Third, the disclosed
antibodies inhibit GDF-8 activity associated with negative
regulation of skeletal muscle mass and bone density. Fourth, the
disclosed antibodies are an effective treatment for muscular
disorders, particularly ALS. These antibodies have many additional
uses, including diagnosing, prognosing, monitoring, screening,
treating, ameliorating, and/or preventing GDF-8-associated
disorders, e.g., muscle and/or bone-associated disorders.
Other aspects of the invention provide compositions comprised of a
GDF-8 antagonist of the invention, e.g., an anti-GDF-8 antibody of
the invention, and the use of such compositions in inhibiting or
neutralizing GDF-8 in animals, particularly in humans or other
animals with muscular disorders such as ALS. The antibodies of the
invention may also be used in a GDF-8-associated disorder, e.g., in
a disorder in which it is desirable to increase muscle tissue or
bone density. For example, anti-GDF-8 antibodies may be used in
therapies and compositions to repair damaged muscle, e.g.,
myocardium, diaphragm, etc. Exemplary GDF-8-associated disorders
and diseases treated by the disclosed methods and compositions
include muscle and neuromuscular disorders such as muscular
dystrophy (including Duchenne's muscular dystrophy); amyotrophic
lateral sclerosis; muscle atrophy; organ atrophy; frailty; tunnel
syndrome; congestive obstructive pulmonary disease (COPD);
sarcopenia, cachexia, and other muscle wasting syndromes; adipose
tissue disorders (e.g., obesity); type 2 diabetes; impaired glucose
tolerance; metabolic syndromes (e.g., syndrome X); insulin
resistance (including resistance induced by trauma, e.g., burns or
nitrogen imbalance), and bone-degenerative diseases (e.g.,
osteoarthritis and osteoporosis). In a preferred, but not limiting,
embodiment of the invention, a composition containing an anti-GDF-8
antibody is used in a method of treating, reducing, or ameliorating
ALS.
In another aspect, the invention provides compositions, e.g.,
pharmaceutical compositions, that include a pharmaceutically
acceptable carrier and at least one GDF-8 antagonist, e.g., an
anti-GDF-8 antibody described herein. In one embodiment, the
compositions, e.g., pharmaceutical compositions, comprise a
combination of two or more of the aforesaid GDF-8 antagonists,
e.g., anti-GDF-8 antibodies or fragments thereof. Also within the
scope of the invention are combinations of the GDF-8 antagonist,
e.g., an anti-GDF-8 antibody, with a therapeutic agent, e.g.,
growth factor inhibitors, immunosuppressants, anti-inflammatory
agents (e.g., systemic anti-inflammatory agents), metabolic
inhibitors, enzyme inhibitors, and/or cytotoxic or cytostatic
agents.
In yet another embodiment, the GDF-8 antagonist, e.g., an
anti-GDF-8 antibody, or a pharmaceutical composition thereof, is
administered alone or in combination therapy, i.e., combined with
other agents, e.g., therapeutic agents, which are useful for
treating GDF-8-associated disorders.
In addition to use in the treatment of various diseases or
disorders, anti-GDF-8 antibodies may be used as diagnostic tools to
quantitatively or qualitatively detect GDF-8 protein or protein
fragments in a biological sample. The presence or amount of GDF-8
protein detected can be correlated with one or more of the medical
conditions listed herein. Thus, in one embodiment, the invention
provides methods to diagnose, prognose, monitor, and/or screen for
GDF-8-associated disorders.
In another aspect, the invention provides a method for detecting
the presence of GDF-8 in a sample in vitro (e.g., a biological
sample, such as serum, plasma, tissue, biopsy). The subject method
can be used to diagnose a GDF-8-associated disorder, e.g., a bone,
muscle, adipose or glucose metabolism-associated disorder. The
method includes: (i) contacting the sample or a control sample with
an anti-GDF-8 antibody as described herein; and (ii) detecting
formation of a complex between the anti-GDF-8 antibody, and the
sample or the control sample, wherein a substantially significant
change in the formation of the complex in the sample relative to
the control sample is indicative of the presence of GDF-8 in the
sample.
In yet another aspect, the invention provides a method for
detecting the presence of GDF-8 in vivo in a subject (e.g., in vivo
imaging in a subject). The subject method can be used to diagnose a
GDF-8-associated disorder, e.g., ALS. The method includes: (i)
administering an anti-GDF-8 antibody as described herein to a
subject or a control subject under conditions that allow binding of
the antibody to GDF-8; and (ii) detecting formation of a complex
between the antibody and GDF-8, wherein a substantially significant
difference in the formation of the complex in the subject relative
to the control subject provides an indication related to the
presence of GDF-8.
Other embodiments of the invention provide a method of diagnosing
or detecting whether a patient is suffering from a GDF-8-associated
disorder (e.g., muscle disorder, neuromuscular disorder, bone
degenerative disorder, metabolic or induced bone disorder, adipose
disorder, glucose metabolism disorder, or insulin-related disorder)
comprising the steps of: (a) obtaining a sample from a patient of
interest; (b) contacting the sample with an anti-GDF-8 antibody as
described herein; (c) determining the level of GDF-8 present in the
patient sample; and (d) comparing the level of GDF-8 in the patient
sample to the level of GDF-8 in a control sample, wherein a
substantial increase, decrease, or similarity in the level of GDF-8
in the patient sample compared to the level of GDF-8 in the control
sample indicates whether the patient is suffering from a
GDF-8-associated disorder.
Another further embodiment of a method for diagnosing or detecting
whether a patient is suffering from a GDF-8-associated disorder
described herein comprises the steps of: (a) obtaining a first
sample taken from the patient of interest; (b) contacting the first
sample with an anti-GDF-8 antibody as described herein; (c)
determining the level of a GDF-8 in the first sample; (d) obtaining
a second sample from an individual not afflicted with the
GDF-8-associated disorder; (e) contacting the second sample with an
anti-GDF-8 antibody as described herein; (f) determining the level
of GDF-8 in the second sample; and (g) comparing the levels of
GDF-8 in the first and second samples, wherein a substantial
increase, decrease, or similarity in the level of first sample
compared to the second sample indicates whether the patient is
suffering from a GDF-8-associated disorder caused (in part or in
full) by overexpression of GDF-8. For example, an increase in the
level of GDF-8 in the first sample compared to the second sample
may indicate that the patient is suffering from the
GDF-8-associated disorder. In contrast, a decrease or similarity in
the level of GDF-8 in the first sample compared to the second
sample may indicate that the patient is not suffering from the
GDF-8-associated disorder.
Antibodies of the invention are also useful in methods of
prognosing the likelihood that a patient will develop a
GDF-8-associated disorder, e.g., a muscle disorder, neuromuscular
disorder, bone degenerative disorder, metabolic or induced bone
disorder, adipose disorder, glucose metabolism disorder, or
insulin-related disorder. In a preferred, but nonlimiting,
embodiment, the method comprises the steps of: (a) obtaining a
first sample from a patient of interest; (b) contacting the first
sample with an anti-GDF-8 antibody as described herein; (c)
determining the level of GDF-8 in the first sample; (d) obtaining a
second sample from an individual not afflicted with the
GDF-8-associated disorder; (e) contacting the second sample with an
anti-GDF-8 antibody as described herein; (f) determining the level
of GDF-8 in the second sample; and (g) comparing the levels of
GDF-8 in the first and second samples, wherein an increase,
decrease, or similarity in the level of GDF-8 in the first sample
as compared with the second sample indicates the likelihood that
the patient will develop the GDF-8-associated disorder. For
example, for a GDF-8-associated disorder caused (in part or in
full) by overexpression of GDF-8, it is likely that the patient
will develop the GDF-8-associated disorder if the first sample has
an increased level of GDF-8 compared to second sample. In contrast,
for a GDF-8-associated disorder caused (in part or in full) by
overexpression of GDF-8, it is unlikely that the patient will
develop the GDF-8-associated disorder if the first sample has a
similar or decreased level of GDF-8 compared to second sample.
Antibodies of the invention are also useful in methods of
monitoring the severity of a GDF-8-associated disorder, e.g.,
muscle disorder, neuromuscular disorder, bone degenerative
disorder, metabolic or induced bone disorder, adipose disorder,
glucose metabolism disorder, or insulin-related disorder. In a
preferred, but not limiting, embodiment, the method comprises the
steps of: (a) obtaining a first sample taken from a patient of
interest at a first time point; (b) contacting the first sample
with an anti-GDF-8 antibody as described herein; (c) determining
the level of GDF-8 in the first sample; (d) obtaining a second
sample taken from the patient at a second time point; (e)
contacting the second sample with an anti-GDF-8 antibody as
described herein; (f) determining the level of GDF-8 in the second
sample; and (g) comparing the levels of GDF-8 in the first and
second samples, wherein an increase, decrease, or similarity in the
level of GDF-8 in the second sample indicates whether the
GDF-8-associated disorder has changed in severity. In one
embodiment, a method of monitoring of the invention is used to
monitor ALS, and a decrease in the level of GDF-8 in the second
sample indicates that ALS has decreased in severity.
An additional method of monitoring a disorder as described herein
comprises the steps of: (a) obtaining a first sample from a patient
of interest; (b) contacting the first sample with an anti-GDF-8
antibody as described herein; (c) determining the level of GDF-8 in
the first sample; (d) obtaining a second sample from an individual
not afflicted with a muscle disorder, neuromuscular disorder, bone
degenerative disorder, metabolic or induced bone disorder, adipose
disorder, glucose metabolism disorder, or insulin-related disorder;
(e) contacting the second sample with an anti-GDF-8 antibody as
described herein; (f) determining the level of GDF-8 in the second
sample; and (g) comparing the levels of GDF-8 in the first and
second samples, wherein an increase, decrease, or similarity in the
level of GDF-8 in the first sample compared to the second sample
indicates the severity of the GDF-8 disorder at that point. In one
embodiment, a method of monitoring of the invention is used to
monitor ALS, and a decrease or similarity in the level of GDF-8 in
the first sample compared to the second sample indicates that ALS
has low severity.
Preferably, the antibody is directly or indirectly labeled with a
detectable substance to facilitate detection of the bound or
unbound antibody. Suitable detectable substances include various
enzymes, prosthetic groups, fluorescent materials, luminescent
materials and radioactive materials.
Methods for delivering or targeting an antagonist of the invention,
e.g., an antibody, to a GDF-8-expressing cell in vivo are also
disclosed herein and are within the scope of the invention.
Kits comprising the GDF-8 antagonists, e.g., the anti-GDF-8
antibodies, of the invention for therapeutic and diagnostic uses
are also within the scope of the invention.
Additional objects of the invention will be set forth in the
following description. Various objects, aspects, and advantages of
the invention will be realized and attained by means of the
elements and combinations particularly pointed out in the
claims.
It is to be understood that both the foregoing general description
and the following detailed description are exemplary and
explanatory only, and are not restrictive of the invention as
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A-C. Characterization of the RK35 anti-GDF-8 antibody. FIG.
1A. Direct binding of RK35 antibody to GDF-8, as measured in an
ELISA assay with biotinylated GDF-8. The binding affinity of RK35
antibody (circles) for GDF-8 was determined to be 4 nM. Control IgG
shows no appreciable binding (squares). FIG. 1B. Effect of RK35
antibody on GDF-8 binding to its high affinity receptor. In a
competition ELISA using the high affinity GDF-8 receptor, ActRIIB
was used to measure the GDF-8 inhibitory activity of RK35. The
binding of biotinylated GDF-8 to immobilized human chimeric protein
ActRIIB fused to the human IgG constant region (Fc) was evaluated
in the absence (diamonds) or presence of various concentrations of
RK35 mAb, soluble ActRIIB or control IgG. Soluble ActRIIB-Fc
receptor (squares) and irrelevant mouse IgG (triangles) were used
as positive and negative controls, respectively. RK35 (circles)
blocked the binding of biotinylated GDF-8 to immobilized ActRIIB
with an IC.sub.50.about.2.5 nM. FIG. 1C. Inhibition of
GDF-8-induced signal transduction. Rhabdomyosarcoma cells
expressing a TGF-.beta. promoter-luciferase fusion gene were
treated with 10 ng/ml of GDF-8 in the absence (squares) or presence
(circles) of varying concentrations of RK35 antibody. RK35 reduced
the GDF-8 induction of luciferase activity in a dose-responsive
manner, with an IC.sub.50 of 0.2 nM. Background (diamonds) signal
was measured with no GDF-8 added.
FIGS. 2A-F. Inhibition of myostatin leads to increased body weight
and increased muscle mass in both SODG93 A mice and rats. FIG. 2A.
Body weights of RK35-treated (squares) (n=11) and PBS-treated
(diamonds) (n=11) transgenic SODG93A mice and PBS-treated
littermate control (wild type) mice (triangles) (n=9). FIG. 2B.
Body weights of male (circles) and female (triangles) RK35-treated
and male (squares) and female (diamonds) PBS-treated transgenic
SODG93A rats (n=10 per group). FIG. 2C. Muscle mass of RK35- and
PBS-treated SODG93A and PBS-treated littermate control mice
(n=9-12) during early-stage disease. Wet weights were determined
for the gastrocnemius (gastroc), cranial tibialis (tibialis),
quadriceps (quad) and diaphragm (diaphragm) muscles from 88-day old
wild type mice treated with PBS (black bars), SODG93A mice treated
with PBS (white bars), and SODG93A mice treated with RK35 (grey
bars). FIG. 2D. Muscle mass from SODG93A rats, treated with PBS
(white bars) or RK35 (grey bars), at early-stage disease (.about.95
days) (n=7 per group). FIG. 2E. Muscle mass of wild type mice
treated with PBS (black bars), SODG93A mice treated with PBS (white
bars), and SODG93A mice treated with RK35 (grey bars) at end-stage
disease (.about.134 days). FIG. 2F. Muscle mass from SODG93A rats,
treated with PBS (white bars) or RK35 (grey bars) at end-stage
disease (.about.128 days). Asterisks (*) denote statistically
(p<0.05) differences between indicated groups.
FIG. 3A-3C. Myostatin inhibition enhances muscle strength in
SODG93A mice and rats. FIG. 3A. Hindlimb grip strength in
PBS-treated wild type mice (triangles), and SODG93A mice treated
with either PBS (diamonds) or RK35 (squares) as a function of age.
Hind limb grip strength is expressed as compression in kilograms
(kg). FIG. 3B. Forelimb grip strength in PBS-treated wild type mice
(triangles), and SODG93A mice treated with either PBS (diamonds) or
RK35 (squares) as a function of age. FIG. 3C. Forelimb grip
strength in wild type rats treated with PBS (WT+PBS), or SODG93A
rats treated with PBS (SOD+PBS) or RK35 (SOD+RK35). For rats,
measurements were taken during a 4-week interval corresponding to
early disease phase, between 95-110 days in age. Forelimb grip
strength is expressed as tension in kilograms (kg). Asterisks (*)
denote a statistically significant difference (p<0.0001) between
indicated groups.
FIG. 4A-K. Effects of myostatin inhibition on muscle structure and
function in SODG93A rodents. Hematoxylin and eosin staining of
medial gastrocnemius muscle from mice at 88 days indicates
significant atrophy in (FIG. 4B) PBS-treated SODG93A mice, in
comparison to either (FIG. 4A) wild type or (FIG. 4C) RK35-treated
SODG93A mice. Hematoxylin and eosin staining of medial
gastrocnemius muscle from both (FIG. 4E) PBS-treated and (FIG. 4F)
RK35-treated SODG93A mice at end-stage indicates both significant
muscle atrophy and centrally placed nuclei (arrowheads) compared to
(FIG. 4D) wild type mouse gastrocnemius. Hematoxylin and eosin
staining of diaphragm from (FIG. 4G) PBS-treated wild type mice and
(FIG. 4H) PBS- or (FIG. 4I) RK35-treated SODG93A mice,
respectively, at end-stage. Examples of atrophic myofibers are
marked ("a"). The asterisk in panel H denotes fiber splitting. Bars
shown denote 50 .mu.m in scale in panels A-F and 25 .mu.m in panels
G-I. Panel FIG. 4J: EMG interference pattern showing inspiratory
bursts, recorded from the diaphragm muscles of wild type rats
treated with PBS (WT+PBS) or SODG93A rats treated with PBS
(SOD+PBS) or RK35 (SOD+RK35). Panel FIG. 4K: EMG burst spike rates
(in Hz) from the diaphragm muscles of wild type rats (n=4), and
from SODG93A rats treated either with vehicle (PBS, n=9) or RK35
(n=8). Asterisks (*) denote a statistically significant difference
(p<0.05) between the indicated groups.
FIG. 5A-D. RK35 treatment slows the decrease in muscle fiber
diameter in gastrocnemius muscle through early-stage disease in
SODG93A mice, and in diaphragm through end-stage disease. Fiber
diameters were measured by morphometry on gastrocnemius muscle from
PBS-treated (FIG. 5A) and RK35-treated (FIG. 5B) SODG93A mice and
PBS-treated wild type mice (FIG. 5C) at 88 days. Means were
significantly different by ANOVA (p<0.0001); pairwise
comparisons by Tukey's multiple comparison post-test were also
significant (p<0.001). By end-stage, no significant differences
in fiber distribution were observed in the gastrocnemius muscle of
PBS-treated and RK35-treated SODG93A mice (data not shown). FIG.
5D. Analysis of fiber diameters of diaphragm muscle from end-stage
PBS-treated and RK35-treated SODG93A mice in comparison to
age-matched wild type control mice. Diaphragm muscle from
RK35-treated SODG93A mice shows a fiber diameter distribution
intermediate between PBS-treated SODG93A mice and wild type control
mice at end-stage. Means were significantly different by ANOVA
(p<0.0001); pairwise comparisons by Tukey's multiple comparison
post-test were also significant (p<0.01). Three muscles per
group were analyzed; linear measurements of the maximum diameter of
the minor axis of at least two hundred fibers were taken, using
Zeiss Axiovision software. Fiber diameters were binned in 20 .mu.m
intervals, and frequency histograms were generated for each muscle
group.
FIG. 6A-J. Effect of anti-myostatin treatment on motor neuron loss
in the ventral horn of the spinal cord. Shown are stereological
analyses of large motor neurons (area greater than 300
(.mu.m.sup.2) from the L3-5 regions of the ventral horn from
SODG93A mice treated with either PBS (SOD+PBS) or RK35 (SOD+RK35)
at early-stage (FIG. 6A) and end-stage (FIG. 6C) disease in
comparison to age-matched wild type mice (WT+PBS). RK35 treatment
showed a trend towards reversing the motor neuron loss (p=0.08) in
early-stage disease (FIG. 6A). Individual counts of large healthy
motor neurons with visible nucleoli were performed on NISSL-stained
sections L3-5 from SODG93A mice treated with either PBS or RK35 at
(FIG. 6B) early-stage and (FIG. 6D) end-stage disease in comparison
to age-matched wild type mice. For each section, both ventral horns
were counted (total of 20 ventral horns per animal) and data are
represented as average number of large motor neurons per ventral
horn. Asterisks (*) denote statistically significant differences
(P<0.001) between the indicated groups. Representative images
from NISSL-stained ventral horn sections (20.times. magnification)
are shown for (FIG. 6E and FIG. 6H) wild type mice treated with
PBS, (FIG. 6F and FIG. 6I) SODG93A mice treated with PBS, and (FIG.
6G and FIG. 6J) SODG93A mice treated with RK35 analyzed at early
(88 days) (FIG. 6E-FIG. 6G) and end-stage disease (134 days; FIG.
6H-FIG. 6J). Bar denotes scale of 200 .mu.m.
FIG. 7 is an image of a dot blot showing epitope mapping of GDF-8
for the RK35 antibody. The binding sites on GDF-8 for RK35 were
identified using forty-eight (48) overlapping 13 amino acid
peptides (13-mers) of human GDF-8 (RK35 antibody binding to 13-mers
1 through 24 is shown in the top row of the blot and RK35 binding
to 13-mers 25-48 is shown in the bottom row of the blot). FIG. 7
discloses amino acid residues 1-55 (top row) and amino acid
residues 56-109 (bottom row) of human GDF-8 relative to SEQ ID NO:
1. The amino acid residue RK35 contact sites with GDF-8 are
indicated in bold.
FIG. 8. Alignment of the light chain and heavy chain variable
regions of RK35 (VL and VH, respectively) with the human germline
frameworks DPK9 and DP-47, respectively. The alignment of the amino
acid sequences of the VL regions of murine RK35 (MRK35 VL: SEQ ID
NO:5). humanized RK35 (HuRK35 VL: SEQ ID NO:9) and qermline DPK-9
(SEQ ID NO:32) is shown. The alignment of the amino acid sequences
of the VH regions of murine RK35 (mRK35 VH: SEQ ID NO:3), humanized
RK35 (HuRK35 VH: SEQ ID NO:7) and qermline DP-47 (SEQ ID NO:33) is
also shown. The amino acids of the murine RK35 (MRK35) variable
chains that are changed in the humanized RK35 (HuRK35) regions are
designated with an asterisk (*) and are in bold; complementarity
determining regions of RK35 are boxed and underlined.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
"Antibody," as used herein, refers to an immunoglobulin or a part
thereof, and encompasses any polypeptide comprising an
antigen-binding site regardless of the source, species of origin,
method of production, and characteristics. For the purposes of the
present invention, it also includes, unless otherwise stated,
antibody fragments such as Fab, F(ab').sub.2, Fv, scFv, Fd, dAb,
diabodies, and other antibody fragments that retain antigen-binding
function. Antibodies can be made, for example, via traditional
hybridoma techniques, recombinant DNA methods, or phage display
techniques using antibody libraries. For various other antibody
production techniques, see Antibodies: A Laboratory Manual, eds.
Harlow et al., Cold Spring Harbor Laboratory, 1988.
The term "antigen-binding domain" refers to the part of an antibody
molecule that comprises the area specifically binding to or
complementary to a part or all of an antigen. Where an antigen is
large, an antibody may only bind to a particular part of the
antigen. The "epitope" or "antigenic determinant" is a portion of
an antigen molecule that is responsible for interactions with the
antigen-binding domain of an antibody. An antigen-binding domain
may be provided by one or more antibody variable domains (e.g., a
so-called Fd antibody fragment consisting of a VH domain). An
antigen-binding domain may comprise an antibody light chain
variable region (VL) and an antibody heavy chain variable region
(VH).
The term "GDF-8" refers to a specific growth and differentiation
factor-8, but not other factors that are structurally or
functionally related to GDF-8, for example, BMP-11 and other
factors belonging to the TGF-.beta. superfamily. The term refers to
the full-length unprocessed precursor form of GDF-8 as well as the
mature and propeptide forms resulting from post-translational
cleavage. The term also refers to any fragments and variants of
GDF-8 that maintain at least some biological activities associated
with mature GDF-8, as discussed herein, including sequences that
have been modified. The amino acid sequence of mature human GDF-8
is provided in SEQ ID NO:1. The present invention relates to GDF-8
from all vertebrate species, including, but not limited to, human,
bovine, chicken, mouse, rat, porcine, ovine, turkey, baboon, and
fish (for sequence information, see, e.g., McPherron et al.,
supra).
The term "GDF-8 activity" refers to one or more of physiologically
growth-regulatory or morphogenetic activities associated with
active GDF-8 protein. For example, active GDF-8 is a negative
regulator of skeletal muscle mass. Active GDF-8 can also modulate
the production of muscle-specific enzymes (e.g., creatine kinase),
stimulate myoblast proliferation, and modulate preadipocyte
differentiation to adipocytes. Exemplary procedures for measuring
GDF-8 activity in vivo and in vitro are set forth in the
Examples.
The term "GDF-8 antagonist" or "GDF-8 inhibitor" includes any agent
capable of inhibiting activity, expression, processing, or
secretion of GDF-8. Such inhibitors include macromolecules and
small molecules, e.g., proteins, antibodies, peptides,
peptidomimetics, siRNA, ribozymes, antisense oligonucleotides,
double-stranded RNA, and other small molecules, that inhibit GDF-8.
A GDF-8 antagonist includes, in addition to the antibodies provided
herein, any antibody that efficiently inhibits GDF-8, including
antibodies with high specificity for binding to GDF-8 (e.g.,
antibodies with a low affinity for other members of the TGF-.beta.
superfamily (e.g., BMP-11)). Variants, including humanized
variants, of these antibodies are contemplated in the methods of
diagnosing, prognosing, monitoring, treating, ameliorating, and
preventing of the invention. Such inhibitors are said to "inhibit,"
"decrease," or "reduce" the biological activity of GDF-8.
The terms "neutralize," "neutralizing," and their cognates refer to
a dramatic reduction or abrogation of GDF-8 activity relative to
the activity of GDF-8 in the absence of the same inhibitor. For
example, a reduction of 75-100% of activity may be said to
"neutralize" GDF-8 activity.
The term "treatment" is used interchangeably herein with the term
"therapeutic method" and refers to both therapeutic treatment and
prophylactic/preventative measures. Those in need of treatment may
include individuals already having a particular medical disorder as
well as those who may ultimately acquire the disorder (i.e., those
needing preventive measures).
The term "isolated" refers to a molecule that is substantially
separated from its natural environment. For instance, an isolated
protein is one that is substantially separated from the cell or
tissue source from which it is derived.
The term "purified" refers to a molecule that is substantially free
of other material that associates with the molecule in its natural
environment. For instance, a purified protein is substantially free
of the cellular material or other proteins from the cell or tissue
from which it is derived. The term refers to preparations where the
isolated protein is sufficiently pure to be administered as a
therapeutic composition, or at least 70% to 80% (w/w) pure, more
preferably, at least 80%-90% (w/w) pure, even more preferably,
90-95% pure; and, most preferably, at least 95%, 96%, 97%, 98%,
99%, or 100% (w/w) pure.
The term "effective dose," "therapeutically effective dose,"
"effective amount," or the like refers to that amount of the
compound that results in either amelioration of symptoms in a
patient or a desired biological outcome (e.g., increasing skeletal
muscle mass and/or bone density). Such amount should be sufficient
to reduce the activity of GDF-8 associated with negative regulation
of skeletal muscle mass and bone density or with glucose
homeostasis and adipose metabolism. The effective amount can be
determined as described herein.
A "disorder associated with GDF-8 activity," "disorder associated
with GDF-8," "GDF-8-associated disorder" or the like refers to
disorders that may be caused, in full or in part, by dysregulation
of (e.g., abnormally increased or decreased) GDF-8 (and/or GDF-8
activity), and/or disorders that may be treated, ameliorated,
prevented, diagnosed, prognosed, or monitored by regulating and/or
monitoring GDF-8 (and/or GDF-8 activity). GDF-8-associated
disorders include muscle disorders, neuromuscular disorders, bone
degenerative disorders, metabolic or induced bone disorders,
adipose disorders, glucose metabolism disorders or insulin-related
disorders. A preferred GDF-8-associated disorder of the invention
is amyotrophic lateral sclerosis (ALS).
The term "small molecule" refers to compounds that are not
macromolecules (see, e.g., Karp (2000) Bioinformatics Ontology
16:269-85; Verkman (2004) AJP-Cell Physiol. 286:465-74). Thus,
small molecules are often considered those compounds that are less
than one thousand daltons (e.g., Voet and Voet, Biochemistry,
2.sup.nd ed., ed. N. Rose, Wiley and Sons, New York, 14 (1995)).
For example, Davis et al. ((2005) Proc. Natl. Acad. Sci. USA
102:5981-86) use the phrase small molecule to indicate folates,
methotrexate, and neuropeptides, while Halpin and Harbury ((2004)
PLos Biology 2:1022-30) use the phrase to indicate small molecule
gene products, e.g., DNAs, RNAs and peptides. Examples of natural
small molecules include, but are not limited to, cholesterols,
neurotransmitters, and siRNAs; synthesized small molecules include,
but are not limited to, various chemicals listed in numerous
commercially available small molecule databases, e.g., FCD (Fine
Chemicals Database), SMID (Small Molecule Interaction Database),
ChEBI (Chemical Entities of Biological Interest), and CSD
(Cambridge Structural Database) (see, e.g., Alfarano et al. (2005)
Nuc. Acids Res. Database Issue 33:D416-24).
II. Antibodies Against GDF-8 and Antibody Fragments
A. Mouse and Humanized Antibody RK35
The present disclosure provides novel antibodies (e.g., intact
antibodies and antibody fragments) that efficiently bind GDF-8. A
nonlimiting illustrative embodiment of such an antibody is termed
RK35. This exemplary embodiment is provided in the form of mouse
and humanized antibodies, and antibody fragments thereof.
The exemplary antibody of the invention, referred to herein as
"RK35," possesses unique and beneficial characteristics. First,
this antibody and antibody fragments are capable of binding mature
GDF-8 with high affinity. Second, the antibody and antibody
fragments of the invention inhibit GDF-8 activity in vitro and in
vivo as demonstrated, for example, by inhibition of ActRIIB binding
and reporter gene assays. Third, the disclosed antibody and
antibody fragments are useful to treat symptoms associated with a
GDF-8-associated disorder, e.g., muscular disorders, particularly
ALS, as demonstrated, e.g., by increasing muscle mass in treated
mutant SOD mice.
In an exemplary embodiment, GDF-8 antagonists are antibodies that
efficiently bind to GDF-8 and inhibit one or more GDF-8 associated
activities. One of ordinary skill in the art will recognize that
the antibodies of the invention may be used to detect, measure, and
inhibit GDF proteins derived from various species, e.g., those
described in the present specification. The percent identity is
determined by standard alignment algorithms such as, for example,
Basic Local Alignment Tool (BLAST) described in Altschul et al.
(1990) J. Mol. Biol. 215:403-10, the algorithm of Needleman et al.
(1970) J. Mol. Biol. 48:444-53, or the algorithm of Meyers et al.
(1988) Comput. Appl. Biosci. 4:11-17. In general, the antibody and
antibody fragments of the invention can be used with any protein
that retains substantial GDF-8 biological activity and comprises an
amino acid sequence which is at least about 70%, 80%, 90%, 95%, or
more identical to any sequence of at least 100, 80, 60, 40, 20, or
15 contiguous amino acids of the mature form of GDF-8 set forth in
SEQ ID NO:1.
B. Antibody Variable Domains
Intact antibodies, also known as immunoglobulins, are typically
tetrameric glycosylated proteins composed of two light (L) chains
of approximately 25 kDa each, and two heavy (H) chains of
approximately 50 kDa each. Two types of light chain, termed lambda
and kappa, exist in antibodies. Depending on the amino acid
sequence of the constant domain of heavy chains, immunoglobulins
are assigned to five major classes: A, D, E, G, and M, and several
of these may be further divided into subclasses (isotypes), e.g.,
IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. Each light chain is composed of an N-terminal variable
(V) domain (VL) and a constant (C) domain (CL). Each heavy chain is
composed of an N-terminal V domain (VH), three or four C domains
(CHs), and a hinge region. The CH domain most proximal to VH is
designated CHI. The VH and VL domains consist of four regions of
relatively conserved sequences named framework regions (FR1, FR2,
FR3, and FR4), which form a scaffold for three regions of
hypervariable sequences (complementarity determining regions,
CDRs). The CDRs contain most of the residues responsible for
interactions of the antibody with the antigen. CDRs are referred to
as CDR1, CDR2, and CDR3. Accordingly, CDR constituents on the heavy
chain are referred to as H1, H2, and H3, while CDR constituents on
the light chain are referred to as L1, L2, and L3. CDR3 is the
greatest source of molecular diversity within the antibody-binding
site. H3, for example, can be as short as two amino acid residues
or greater than 26 amino acids. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known in the art. For a review of the
antibody structure, see Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, eds. Harlow et al., 1988. One of skill in
the art will recognize that each subunit structure, e.g., a CH, VH,
CL, VL, CDR, and/or FR structure, comprises active fragments. For
example, active fragments may consist of the portion of the VH, VL,
or CDR subunit that binds the antigen, i.e., the antigen-binding
fragment, or the portion of the CH subunit that binds to and/or
activates an Fc receptor and/or complement.
Nonlimiting examples of binding fragments encompassed within the
term "antibody fragment" used herein include: (i) an Fab fragment,
a monovalent fragment consisting of the VL, VH, CL and CH1 domains;
(ii) an F(ab').sub.2 fragment, a bivalent fragment comprising two
Fab fragments linked by a disulfide bridge at the hinge region;
(iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an
Fv fragment consisting of the VL and VH domains of a single arm of
an antibody, (v) a dAb fragment, which consists of a VH domain; and
(vi) an isolated CDR. Furthermore, although the two domains of the
Fv fragment, VL and VH, are coded for by separate genes, they may
be recombinantly joined by a synthetic linker, creating a single
protein chain in which the VL and VH regions pair to form
monovalent molecules (known as single chain Fv (scFv). The most
commonly used linker is a 15-residue (Gly4Ser).sub.3 (SEQ ID NO:
34) peptide, but other linkers are also known in the art. Single
chain antibodies are also intended to be encompassed within the
term "antibody" or "antigen-binding fragment" of an antibody. These
antibodies are obtained using conventional techniques known to
those skilled in the art, and the fragments are screened for
utility in the same manner as intact antibodies.
Antibody diversity is created by multiple germline genes encoding
variable regions and a variety of somatic events. The somatic
events include recombination of variable gene segments with
diversity (D) and joining (J) gene segments to make a complete VH
region, and the recombination of variable and joining gene segments
to make a complete VL region. The recombination process itself is
imprecise, resulting in the loss or addition of amino acids at the
V(D)J junctions. These mechanisms of diversity occur in the
developing B cell prior to antigen exposure. After antigenic
stimulation, the expressed antibody genes in B cells undergo
somatic mutation. Based on the estimated number of germline gene
segments, the random recombination of these segments, and random
VH-VL pairing, up to 1.6.times.10.sup.7 different antibodies may be
produced (Fundamental Immunology, 3rd ed. (1993), ed. Paul, Raven
Press, New York, N.Y.). When other processes that contribute to
antibody diversity (such as somatic mutation) are taken into
account, it is thought that upwards of 1.times.10.sup.10 different
antibodies may be generated (Immunoglobulin Genes, 2nd ed. (1995),
eds. Jonio et al., Academic Press, San Diego, Calif.). Because of
the many processes involved in generating antibody diversity, it is
unlikely that independently derived monoclonal antibodies with the
same antigen specificity will have identical amino acid
sequences.
Thus, the present invention provides novel antibodies that bind
GDF-8. The antibody fragments of the invention, e.g., structures
containing a CDR, will generally be an antibody heavy or light
chain sequence, or an active fragment thereof, in which the CDR is
placed at a location corresponding to the CDR of naturally
occurring VH and VL. The structures and locations of immunoglobulin
variable domains, e.g., CDRs, may be defined using well-known
numbering schemes, e.g., the Kabat numbering scheme, the Chothia
numbering scheme, a combination of Kabat and Chothia (AbM), etc.
(see, e.g., Sequences of Proteins of Immunological Interest, U.S.
Department of Health and Human Services (1991), eds. Kabat et al.;
Al-Lazikani et al. (1997) J. Mol. Biol. 273:927-48).
Thus, the present invention further provides novel CDRs. The
structure for carrying a CDR of the invention will generally be a
polypeptide, e.g., an antibody heavy or light chain sequence or a
substantial portion thereof, in which the CDR is located at a
position corresponding to the CDR of naturally occurring VH and VL
regions. The structures and locations of immunoglobulin variable
domains may be determined as described in, e.g., Kabat et al.,
supra and Al-Lazikani et al., supra.
Antibody molecules (including antibody fragments) of the present
invention, i.e., antibody molecules that antagonize GDF-8, include,
but are not limited to, murine monoclonal antibody RK35 and its
variants, specifically the humanized variant. GDF-8 antagonists of
the invention include, in addition to RK35, other antibodies that
bind efficiently to GDF-8, including antibodies with high
specificity for binding to GDF-8 (e.g., antibodies with a lower
affinity for other members of the TGF-.beta. superfamily (e.g.,
BMP-11)). Variants, including humanized variants, of these
antibodies are contemplated in the methods of diagnosing,
prognosing, monitoring, treating, ameliorating, and preventing of
the invention. These antibody molecules may be useful in preventing
or treating a GDF-8-associated disorder, e.g., bone, muscle,
adipose and glucose metabolism-related pathologies. The amino acid
sequences of the light chain variable regions of murine and
humanized RK35 are set forth in SEQ ID NOs:5 and 9, respectively.
The amino acid sequences of the heavy chain variable regions of
murine and humanized RK35 are set forth in SEQ ID NOs:3 and 7,
respectively. The amino acid sequences of the three complementarity
determining regions (CDRs) in the variable light chains of murine
and humanized RK35 are set forth in SEQ ID NOs:13, 14, 15, 23, 24,
and 25. The amino acid sequences of the three CDRs in the variable
heavy chains of murine and humanized RK35 are set forth in SEQ ID
NOs:10, 11, 12, 20, 21, and 22.
As described above, the CDRs contain most of the residues
responsible for interactions with an antigen, and are contained
within the VH and VL domains, i.e., the heavy chain variable region
and the light chain variable region, respectively. Consequently,
provided that an antibody comprises at least one CDR comprising an
amino acid sequence selected from the amino acid sequences set
forth in SEQ ID NOs:10-15 and 20-25, or an active antibody fragment
thereof, it is an antibody of the invention, i.e., one that binds
to GDF-8 and interferes with GDF-8 signaling. Therefore, an
embodiment of the invention includes polypeptides, e.g.,
antibodies, that contain one or more CDRs that comprise an amino
acid sequence selected from the amino acid sequences set forth in
SEQ ID NOs:10-15 and 20-25, or an active fragment thereof.
Consequently, one of skill in the art will recognize that the
antibodies of the invention include an antibody in which the CDRs
of the VL chain are those set forth in SEQ ID NOs:13-15 and 23-25,
or the CDRs of the VH chain are those set forth in SEQ ID NOs:10-12
and 20-22.
An antigen-binding fragment may be an Fv fragment, which consists
of VH and VL domains. Thus, an Fv fragment of RK35 may constitute
an antibody of the invention, provided that it binds to GDF-8 and
interferes with GDF-8 signaling. One of skill in the art will
recognize that any antibody fragment containing the Fv fragment of,
e.g., RK35, may also be an antibody of the invention. Additionally,
any Fv fragment, scFv fragment, Fab fragment, or F(ab').sub.2
fragment, that contains one or more CDRs having an amino acid
sequence selected from the amino acid sequences set forth in SEQ ID
NOs:10-15 and 20-25, may also be an antibody of the invention.
Such antibody molecules may be produced by methods well known to
those skilled in the art. For example, monoclonal antibodies may be
produced by generation of hybridomas in accordance with known
methods. Hybridomas formed in this manner are then screened using
standard methods, such as enzyme-linked immunosorbent assay (ELISA)
and Biacore analysis, to identify one or more hybridomas that
produce an antibody that binds GDF-8, interferes with GDF-8
signaling, and neutralizes or inhibits one or more GDF-8-associated
activities. Recombinant GDF-8, naturally occurring GDF-8, any
variants thereof, and antigenic peptide fragments of GDF-8 may be
used as the immunogen. An antigenic peptide fragment of GDF-8
comprises at least seven continuous amino acid residues and
encompasses an epitope such that an antibody raised against the
peptide forms an immune complex with GDF-8. Preferably, the
antigenic peptide comprises at least 10 amino acid residues, more
preferably at least 15 amino acid residues, even more preferably at
least 20 amino acid residues, and most preferably at least 30 amino
acid residues. Additionally, it is preferable that the antigenic
peptide fragment of GDF-8 comprises the GDF-8 receptor-binding
site.
Polyclonal sera and antibodies of the invention may be produced by
immunizing a suitable subject with GDF-8, its variants, and/or
portions thereof. The antibody titer in the immunized subject may
be monitored over time by standard techniques, such as an ELISA, or
by using immobilized GDF-8 or other marker proteins (e.g., FLAG).
If desired, the antibody molecules of the present invention may be
isolated from the subject or culture media and further purified by
well-known techniques, such as protein A chromatography, to obtain
an IgG fraction.
Certain embodiments of the invention comprise the VH and/or VL
domain of the Fv fragment of RK35. Fragments of antibodies of the
present invention, e.g., Fab, F(ab').sub.2, Fd, and dAb fragments,
may be produced by cleavage of the antibodies in accordance with
methods well known in the art. For example, immunologically active
Fab and F(ab').sub.2 fragments may be generated by treating the
antibodies with an enzyme such as papain and pepsin.
Further embodiments comprise one or more CDRs of any of these VH
and VL domains, as set forth in SEQ ID NOs:10-15 and 20-25. One
embodiment comprises an H3 fragment of the VH domain of RK35 as set
forth in SEQ ID NO:12.
DNA and amino acid (AA) sequences of VH and VL domains, and CDRs of
the presently disclosed antibodies are enumerated as listed in
Table 1. For convenience, the approximate positions of each CDR
within the VH and VL domains are listed in Table 2.
TABLE-US-00001 TABLE 1 DNA and Amino Acid Sequences of VH, VL, CH,
CL and CDRs in RK35 MOUSE DNA seq. of VH SEQ ID NO: 2 AA seq. of VH
SEQ ID NO: 3 DNA seq. of VL SEQ ID NO: 4 AA seq. of VL SEQ ID NO: 5
HUMANIZED DNA seq. of VH SEQ ID NO: 6 AA seq. of VH SEQ ID NO: 7
DNA seq. of VL SEQ ID NO: 8 AA seq. of VL SEQ ID NO: 9 CDRs BASED
AA sequence of SEQ ID NO: 10 or SEQ ID NO: 20 ON KABAT H1 OR AbM
(ital) AA sequence of SEQ ID NO: 11 or SEQ ID NO: 21 DEFINITIONS H2
AA sequence of SEQ ID NO: 12 or SEQ ID NO: 22 H3 AA sequence of SEQ
ID NO: 13 or SEQ ID NO: 23 L1 AA sequence of SEQ ID NO: 14 or SEQ
ID NO: 24 L2 AA sequence of SEQ ID NO: 15 or SEQ ID NO: 25 L3 DNA
seq. of CL SEQ ID NO: 16 AA seq. of CL SEQ ID NO: 17 DNA seq. of CH
SEQ ID NO: 18 AA seq. of CH SEQ ID NO: 19
TABLE-US-00002 TABLE 2 Approximate CDR position according to Kabat
(not ital.) or AbM (ital.) definitions within variable regions of
RK35 mouse and humanized antibodies RK35 RK35 CDR SEQ ID NO: 3 SEQ
ID NO: 7 HI 31-35 or 26-35 31-35 or 26-35 H2 50-66 or 50-59 50-66
or 50-59 H3 99-105 or 99-105 99-105 or 99-105 SEQ ID NO: 5 SEQ ID
NO: 9 LI 24-34 or 24-34 24-34 or 24-34 L2 50-56 or 50-56 50-56 or
50-56 L3 89-95 or 89-95 89-95 or 89-95
Anti-GDF-8 antibodies may further comprise antibody constant
regions or parts thereof. For example, a VL domain of the invention
may be attached at its C-terminal end to an antibody light chain
constant domain, e.g., a human C.kappa. or C.lamda. chain,
preferably a C.lamda. chain. Similarly, an antigen-binding fragment
based on a VH domain may be attached at its C-terminal end to all
or part of an immunoglobulin heavy chain derived from any antibody
isotype, e.g., IgG, IgA, IgE, and IgM, and any of the isotype
subclasses, particularly IgG.sub.1 and IgG.sub.4. In exemplary
embodiments, antibodies comprise C-terminal fragments of heavy and
light chains of human IgG1. Preferred DNA and amino acid sequences
for the C-terminal constant fragment of the light X chain are set
forth in SEQ ID NO:16 and SEQ ID NO:17, respectively. Preferred DNA
and amino acid sequences for the C-terminal constant fragment of
IgG.sub.1 heavy chain are set forth in SEQ ID NO:18 and SEQ ID
NO:19, respectively. It is understood that, due to the degeneracy
of the genetic code, DNA sequences listed in Table 1 are merely
representative of nucleic acids that encode the amino acid
sequences, peptides, and antibodies of interest, and are not to be
construed as limiting.
Certain embodiments of the invention comprise the VH and/or VL
domain of the Fv fragment of RK35. Further embodiments comprise one
or more complementarity determining regions (CDRs) of any of these
VH and VL domains. One embodiment comprises an H3 fragment of the
VH domain of RK35. The VH and VL domains of the invention, in
certain embodiments, are germlined, i.e., the framework regions
(FRs) of these domains are changed using conventional molecular
biology techniques to match the consensus amino acid sequences of
human germline gene products. This is also known as a humanized or
germlined antibody. In other embodiments, the framework sequences
remain diverged from the germline. Humanized antibodies may be
produced using transgenic mice that are incapable of expressing
endogenous immunoglobulin heavy and light chain genes, but are
capable of expressing human heavy and light chain genes.
C. Modified Antibodies and their Fragments
A further aspect of the invention provides methods for obtaining an
antibody antigen-binding domain directed against GDF-8. The skilled
artisan will appreciate that the antibodies of the invention are
not limited to the specific sequences of VH and VL as listed in
Table 1 but also include variants of these sequences that retain
antigen-binding ability. Such variants may be derived from the
provided sequences using techniques known in the art. Amino acid
substitutions, deletions, or additions, can be made in either the
FRs or in the CDRs. While changes in the framework regions are
usually designed to improve stability and reduce immunogenicity of
the antibody, changes in the CDRs are usually designed to increase
affinity of the antibody for its target. Such affinity-increasing
changes are typically determined empirically by altering the CDR
and testing the antibody. Such alterations can be made according to
the methods described in, e.g., Antibody Engineering, 2nd. ed.,
Borrebaeck, ed., Oxford University Press, 1995.
Thus, the antibodies of the invention also include those that bind
to GDF-8, interfere with GDF-8 signaling, and have mutations in the
constant regions of the heavy and light chains. It is sometimes
desirable to mutate and inactivate certain fragments of the
constant region. For example, mutations in the heavy constant
region are sometimes desirable to produce antibodies with reduced
binding to the Fc receptor (FcR) and/or complement; such mutations
are well known in the art. One of skill in the art will also
recognize that the determination of which active fragments of the
CL and CH subunits are necessary will depend on the application to
which an antibody of the invention is applied. For example, the
active fragments of the CL and CH subunits that are involved with
their covalent link to each other will be important in the
generation of an intact antibody.
The method for making a VH domain that is an amino acid sequence
variant of a VH domain set out herein comprises a step of adding,
deleting, substituting or inserting one or more amino acids in the
amino acid sequence of the presently disclosed VH domain,
optionally combining the VH domain thus provided with one or more
VL domains, and testing the VH domain or VH/VL combination or
combinations for binding to GDF-8, and (preferably) testing the
ability of such antigen-binding domain to modulate one or more
GDF-8-associated activities. The VL domain may have an amino acid
sequence that is substantially as set out herein. An analogous
method may be employed in which one or more sequence variants of a
VL domain disclosed herein are combined with one or more VH
domains.
A further aspect of the invention provides a method of preparing an
antigen-binding fragment that interacts with GDF-8. The method
comprises:
(a) providing a starting repertoire of nucleic acids encoding a VH
domain that either includes a CDR, e.g., CDR3, to be replaced or a
VH domain that lacks a CDR, e.g., CDR3, encoding region;
(b) combining the repertoire with a donor nucleic acid encoding a
donor CDR comprising an active fragment of SEQ ID NO:2 or 6, e.g.,
a donor nucleic acid encoding the amino acid sequence set forth in
SEQ ID NO:3 or 7, such that the donor nucleic acid is inserted into
the CDR, e.g., CDR3, region in the repertoire so as to provide a
product repertoire of nucleic acids encoding a VH domain;
(c) expressing the nucleic acids of the product repertoire;
(d) selecting an antigen-binding fragment that interacts with
GDF-8; and
(e) recovering the selected antigen-binding fragment or nucleic
acid encoding it.
Again, an analogous method may be employed in which a VL CDR (e.g.,
L3) of the invention is combined with a repertoire of nucleic acids
encoding a VL domain, which either includes a CDR to be replaced or
lacks a CDR encoding region.
A coding sequence of a CDR of the invention (e.g., CDR3) may be
introduced into a repertoire of variable domains lacking a CDR
(e.g., CDR3), using recombinant DNA technology. For example, Marks
et al. (1992) Bio/Technology 10:779-83, describes methods of
producing repertoires of antibody variable domains in which
consensus primers directed at or adjacent to the 5' end of the
variable domain area are used in conjunction with consensus primers
to the third framework region of human VH genes to provide a
repertoire of VH variable domains lacking a CDR3. The repertoire
may be combined with a CDR3 of a particular antibody. Using
analogous techniques, the CDR3-derived sequences of the present
invention may be shuffled with repertoires of VH or VL domains
lacking a CDR3, and the shuffled complete VH or VL domains combined
with a cognate VL or VH domain to provide antigen-binding fragments
of the invention. The repertoire may then be displayed in a
suitable host system, such as the phage display system of, e.g., WO
92/01047, so that suitable antigen-binding fragments can be
selected.
Analogous shuffling or combinatorial techniques are also disclosed
by Stemmer (1994) Nature 370:389-91, which describes a technique in
relation to a .beta.-lactamase gene but observes that the approach
may be used for the generation of antibodies.
A further alternative is to generate novel VH or VL regions
carrying a CDR-derived sequence of the invention using random
mutagenesis of one or more selected VH and/or VL genes to generate
mutations within the entire variable domain. Such a technique is
described in Gram et al. (1992) Proc. Natl. Acad. Sci. U.S.A.
89:3576-80 by using error-prone PCR.
Another method that may be used to generate novel antibodies or
fragments thereof is to direct mutagenesis to CDRs of VH or VL
genes. Such techniques are disclosed in Barbas et al. (1994) Proc.
Natl. Acad. Sci. U.S.A. 91:3809-13 and Schier et al. (1996) J. Mol.
Biol. 263:551-67.
Similarly, one, two, or all three CDRs, may be grafted into a
repertoire of VH or VL domains which are then screened for a
binding partner or binding fragments for GDF-8.
A substantial portion of an immunoglobulin variable domain will
comprise at least the CDRs and, optionally, their intervening
framework regions from the antibody fragments as set out herein.
The portion will also include at least about 50% of either or both
of FR1 and FR4, the 50% being the C-terminal 50% of FR1 and the
N-terminal 50% of FR4. Additional residues at the N-terminal or
C-terminal end of the substantial part of the variable domain may
be those not normally associated with naturally occurring variable
domain regions. For example, construction of antibody fragments of
the present invention made by recombinant DNA techniques may result
in the introduction of N- or C-terminal residues encoded by linkers
introduced to facilitate cloning or other manipulation steps. Other
manipulation steps include the introduction of linkers to join
variable domains of the invention to further protein sequences
including immunoglobulin heavy chains, other variable domains (for
example, in the production of diabodies) or protein labels as
discussed in more details below.
Although the embodiments illustrated in the Examples comprise a
"matching" pair of VH and VL domains, the invention also
encompasses binding fragments containing a single variable domain,
e.g., a dAb fragment, derived from either VH or VL domain
sequences, especially VH domains. In the case of either of the
single chain binding domains, these domains may be used to screen
for complementary domains capable of forming a two-domain
antigen-binding domain capable of binding GDF-8. This may be
achieved by phage display screening methods using the so-called
hierarchical dual combinatorial approach as disclosed in, e.g., WO
92/01047. In this technique, an individual colony containing either
an H or L chain clone is used to infect a complete library of
clones encoding the other chain (L or H) and the resulting
two-chain antigen-binding domain is selected in accordance with
phage display techniques, such as those described in that
reference. This technique is also disclosed in Marks et al.,
supra.
Antibodies can be conjugated by chemical methods with
radionuclides, drugs, macromolecules, or other agents, and may be
made as fusion proteins comprising one or more CDRs of the
invention.
An antibody fusion protein contains a VH-VL pair in which one of
these chains (usually VH) and another protein are synthesized as a
single polypeptide chain. These types of products differ from
antibodies in that they generally have an additional functional
element--the active moiety of a small molecule or the principal
molecular structural feature of the conjugated or fused
macromolecule.
In addition to the changes to the amino acid sequence outlined
above, the antibodies can be glycosylated, pegylated, or linked to
albumin or a nonproteinaceous polymer. For instance, anti-GDF-8
antibodies may be linked to one of a variety of nonproteinaceous
polymers, e.g., polyethylene glycol, polypropylene glycol, or
polyoxyalkylenes. The antibodies may be chemically modified, e.g.,
to increase their circulating half-life by covalent conjugation to
a polymer. Exemplary polymers, and methods to attach them to
peptides are known in the art.
In other embodiments, the antibody may be modified to have an
altered glycosylation pattern (i.e., relative to the original or
native glycosylation pattern). As used herein, "altered" means
having one or more carbohydrate moieties deleted, and/or having one
or more glycosylation sites added to the original antibody.
Addition of glycosylation sites to an anti-GDF-8 antibody is
accomplished by well-known methods of altering the amino acid
sequence to contain glycosylation site consensus sequences. Another
means of increasing the number of carbohydrate moieties on the
antibodies is by chemical or enzymatic coupling of glycosides to
the amino acid residues of the antibody. Removal of any
carbohydrate moieties present on the antibodies may be accomplished
chemically or enzymatically as known in the art.
Antibodies of the invention may also be tagged with a detectable or
functional label such as .sup.131I or .sup.99Tc, which may be
attached to antibodies of the invention using conventional
chemistry known in the art. Labels also include enzyme labels such
as horseradish peroxidase or alkaline phosphatase. Labels further
include chemical moieties such as biotin, which may be detected via
binding to a specific cognate detectable moiety, e.g., labeled
avidin.
Antibodies, in which CDR sequences differ only insubstantially from
those listed in Table 1, are encompassed within the scope of the
invention. Insubstantial differences include minor amino acid
changes, e.g., substitutions of one or two out of any five amino
acids in the sequence of a CDR. Typically, an amino acid is
substituted by a related amino acid having similar charge,
hydrophobicity, or stereochemical characteristics. Such
substitutions would be within the ordinary skills of an artisan.
The structure framework regions (FRs) can be modified more
substantially than CDRs without adversely affecting the binding
properties of an antibody. Changes to FRs include, but are not
limited to, humanizing a nonhuman derived framework or engineering
certain framework residues that are important for antigen contact
or for stabilizing the binding site, e.g., changing the class or
subclass of the constant region, changing specific amino acid
residues which might alter an effector function such as Fc receptor
binding (e.g., Lund et al. (1991) J. Immunol. 147:2657-62; Morgan
et al. (1995) Immunology 86:319-24), or changing the species from
which the constant region is derived. Antibodies may have mutations
in the CH2 region of the heavy chain that reduce or alter effector
function, e.g., Fc receptor binding and complement activation. For
example, antibodies may have mutations such as those described in
U.S. Pat. Nos. 5,624,821 and 5,648,260. In the IgG.sub.1 or
IgG.sub.2 heavy chain, for example, such mutations may be made at
amino acid residues 117 and 120 of SEQ ID NO:19, which represents
the Fc portion of IgG.sub.1 (these residues correspond to amino
acids 234 and 237 in the full-length sequence of IgG.sub.1 or
IgG.sub.2). Antibodies may also have mutations that stabilize the
disulfide bond between the two heavy chains of an immunoglobulin,
such as mutations in the hinge region of IgG.sub.4, as disclosed
in, e.g., Angal et al. (1993) Mol. Immunol. 30:105-08.
The polypeptides and antibodies of the present invention also
encompass proteins that are structurally different from the
disclosed polypeptides and antibodies, e.g., which have an altered
sequence but substantially the same biochemical properties as the
disclosed polypeptides and antibodies, e.g., have changes only in
functionally nonessential amino acids. Such molecules include
naturally occurring allelic variants and deliberately engineered
variants containing alterations, substitutions, replacements,
insertions, or deletions. Techniques for such alterations,
substitutions, replacements, insertions, or deletions are well
known to those skilled in the art.
Antibodies of the invention may additionally be produced using
transgenic nonhuman animals that are modified so as to produce
fully human antibodies rather than the animal's endogenous
antibodies in response to challenge by an antigen. See, e.g., PCT
publication WO 94/02602. The endogenous genes encoding the heavy
and light immunoglobulin chains in the nonhuman host have been
incapacitated, and active loci encoding human heavy and light chain
immunoglobulins are inserted into the host's genome. The human
genes are incorporated, for example, using yeast artificial
chromosomes containing the requisite human DNA segments. An animal
which provides all the desired modifications is then obtained as
progeny by crossbreeding intermediate transgenic animals containing
fewer than the full complement of the modifications. One embodiment
of such a nonhuman animal is a mouse, and is termed the
XENOMOUSE.TM. as disclosed in PCT publications WO 96/33735 and WO
96/34096. This animal produces B cells that secrete fully human
immunoglobulins. The antibodies can be obtained directly from the
animal after immunization with an immunogen of interest, as, for
example, a preparation of a polyclonal antibody, or alternatively
from immortalized B cells derived from the animal, such as
hybridomas producing monoclonal antibodies. Additionally, the genes
encoding the immunoglobulins with human variable regions can be
recovered and expressed to obtain the antibodies directly, or can
be further modified to obtain analogs of antibodies such as, for
example, single chain Fv molecules.
Consequently, the term antibody as used herein includes intact
antibodies, fragments of antibodies, e.g., Fab, F(ab').sub.2, Fd,
dAb and scFv fragments, and intact antibodies and fragments that
have been mutated either in their constant and/or variable regions
(e.g., mutations to produce chimeric, partially humanized, or fully
humanized antibodies, as well as to produce antibodies with a
desired trait, e.g., enhanced GDF-8 binding and/or reduced FcR
binding). As such these antibodies are included in the scope of the
invention, provided that the antibody binds specifically to GDF-8,
interferes with GDF-8 signaling, and/or neutralizes or inhibits one
or more GDF-8-associated activities.
Other protein-binding molecules may also be employed to modulate
the activity of GDF-8. Such protein-binding molecules include small
modular immunopharmaceutical (SMIP.TM.) drugs (Trubion
Pharmaceuticals, Seattle, Wash.). SMIPs are single-chain
polypeptides composed of a binding domain for a cognate structure
such as an antigen, a counterreceptor or the like, a hinge-region
polypeptide having either one or no cysteine residues, and
immunoglobulin CH2 and CH3 domains (see also www.trubion.com).
SMIPs and their uses and applications are disclosed in, e.g., U.S.
Published Patent Appln. Nos. 2003/0118592, 2003/0133939,
2004/0058445, 2005/0136049, 2005/0175614, 2005/0180970,
2005/0186216, 2005/0202012, 2005/0202023, 2005/0202028,
2005/0202534, and 2005/0238646, and related patent family members
thereof, all of which are hereby incorporated by reference herein
in their entireties.
The binding capacity of an antibody of the invention may be
measured by the following methods: Biacore analysis, enzyme linked
immunosorbent assay (ELISA), X-ray crystallography, sequence
analysis and scanning mutagenesis as described in the Examples
below, and other methods that are well known in the art. The
ability of an antibody of the invention to neutralize and/or
inhibit one or more GDF-8-associated activities may be measured by
the following nonlimiting list of methods: assays for measuring the
proliferation of a GDF-8-dependent cell line; assays for measuring
the expression of GDF-8-mediated polypeptides; assays measuring the
activity of downstream signaling molecules; assays testing the
efficiency of an antibody of the invention to prevent muscle
disorders in a relevant animal model; assays as described in the
Examples below; and other assays that are well known in the
art.
A further aspect of the invention provides a method of selecting
antibodies capable of binding GDF-8 and neutralizing and/or
inhibiting one or more GDF-8-associated activities. The method
comprises:
a) contracting a plurality of antibodies with GDF-8;
b) choosing antibodies that bind to GDF-8;
c) testing the ability of chosen antibodies to prevent GDF-8 from
binding to the GDF-8 receptor; and
d) selecting antibodies capable of preventing GDF-8 from binding to
its receptor.
The anti-GDF-8 antibodies of the invention are also useful for
isolating, purifying, and/or detecting GDF-8 in supernatants,
cellular lysates, or on a cell surface. Antibodies disclosed in
this invention can be used diagnostically to monitor GDF-8 protein
levels as part of a clinical testing procedure. Additionally,
antibodies of the invention can be used in treatments requiring the
neutralization and/or inhibition of one or more GDF-8-associated
activities, e.g., treatments for ALS and other muscle-related
pathologies. The present invention also provides novel isolated and
purified polynucleotides and polypeptides related to novel
antibodies directed against human GDF-8. The genes,
polynucleotides, proteins, and polypeptides of the present
invention include, but are not limited to, murine and humanized
antibodies to GDF-8 (e.g., RK35) and variants thereof.
D. Nucleic Acids, Cloning, and Expression Systems
The present invention further provides isolated and purified
nucleic acids encoding antibodies of the present invention. Nucleic
acids according to the present invention may comprise DNA or RNA
and may be wholly or partially synthetic. Reference to nucleotide
sequences as set out herein encompass DNA molecules with the
specified sequences or genomic equivalents, as well as RNA
molecules with the specified sequences in which U is substituted
for T, unless context requires otherwise.
For example, the invention provides purified and isolated
polynucleotides encoding the variable region of a murine antibody
to GDF-8 that modulates one or more GDF-8-associated activities
(e.g., neutralizes GDF-8 bioactivity) (RK35), and a humanized
version of RK35. Preferred DNA sequences of the invention include
genomic, cDNA, and chemically synthesized DNA sequences.
The nucleotide sequences of the invention include those that encode
the light chain variable regions of mouse RK35 set forth in SEQ ID
NO:4, including those that encode a leader sequence preceding the
light chain variable region sequence, e.g., the nucleotide sequence
set forth as SEQ ID NO:30 (nucleotides 1-60 correspond to the
leader sequence, and nucleotides 61-381 correspond to SEQ ID NO:4).
The nucleotide sequences of the invention also include those that
encode the heavy chain variable region of RK35 set forth in SEQ ID
NO:2, including those that encode a leader sequence preceding the
heavy chain variable region, e.g., the nucleotide sequence set
forth as SEQ ID NO:28 (nucleotides 1-57 correspond to the leader
sequence, and nucleotides 58-405 correspond to SEQ ID NO:2). The
nucleotide sequences of the invention also include humanized
sequences of the heavy and light chain variable regions, such as
those set forth in SEQ ID NOs:6 and 8, respectively.
Polynucleotides of the present invention also include
polynucleotides that hybridize under stringent conditions to the
nucleic acid sequences set forth in SEQ ID NOs:2, 4, 6, and 8, and
complements thereof, and/or encode polypeptides that retain
substantial biological activity (i.e., active fragments) in the
variable regions. Polynucleotides of the present invention also
include continuous portions of the sequences set forth in SEQ ID
NOs:2, 4, 6, and 8, comprising at least 15 consecutive
nucleotides.
The amino acid sequence of the variable light chains of mouse RK35
is set forth in SEQ ID NO:5. An example of an amino acid sequence
of the variable light chain domain of mouse RK35 preceded by a
leader sequence is set forth as SEQ ID NO:31. The amino acid
sequence of the variable heavy chains of RK35 is set forth in SEQ
ID NO:3. An example of an amino acid sequence of the variable heavy
chain domain of mouse RK35 preceded by a leader sequence is set
forth as SEQ ID NO:29. The amino acid sequences of humanized
variable heavy and light chains are set out in SEQ ID NOs:7 and 9,
respectively. The amino acid sequences of the CDRs contained within
the heavy chains of mouse RK35 are set forth in SEQ ID NOs:10-12
and 20-22. The amino acid sequences of the CDRs contained within
the light chains of mouse RK35 are set forth in SEQ ID NOs:13-15
and 23-25. Polypeptides of the present invention also include
continuous portions of any of the sequences substantially set forth
in SEQ ID NOs:3, 5, 7, 9, 10-15, and 20-25 comprising at least 5
consecutive amino acids. A preferred polypeptide of the present
invention includes any continuous portion of any sequence
substantially set forth in SEQ ID NOs:3, 5, 7, 9, and 10-15
retaining substantial biological activity of an antibody of the
invention. In addition to those polynucleotides described above,
the present invention also includes polynucleotides that encode the
amino acid sequences substantially set forth in SEQ ID NOs:3, 5, 7,
9, and 10-15 or a continuous portion thereof, and that differ from
the polynucleotides described above only due to the well-known
degeneracy of the genetic code.
The isolated polynucleotides of the present invention may be used
as hybridization probes and primers to identify and isolate nucleic
acids having sequences identical to or similar to those encoding
the disclosed polynucleotides. Polynucleotides isolated in this
fashion may be used, for example, to produce antibodies against
GDF-8 or other TGF-.beta. family members or to identify cells
expressing such antibodies. Hybridization methods for identifying
and isolating nucleic acids include polymerase chain reaction
(PCR), Southern hybridizations, in situ hybridization and Northern
hybridization, and are well known to those skilled in the art.
Hybridization reactions can be performed under conditions of
different stringencies. The stringency of a hybridization reaction
includes the difficulty with which any two nucleic acid molecules
will hybridize to one another. Preferably, each hybridizing
polynucleotide hybridizes to its corresponding polynucleotide under
reduced stringency conditions, more preferably stringent
conditions, and most preferably highly stringent conditions.
Examples of stringency conditions are shown in Table 3 below:
highly stringent conditions are those that are at least as
stringent as, for example, conditions A-F; stringent conditions are
at least as stringent as, for example, conditions G-L; and reduced
stringency conditions are at least as stringent as, for example,
conditions M-R.
TABLE-US-00003 TABLE 3 Hybrid Hybridization Wash Length Temperature
and Temperature Condition Hybrid (bp).sup.1 Buffer.sup.2 and
Buffer.sup.2 A DNA:DNA >50 65.degree. C.; 1X SSC -or- 65.degree.
C.; 0.3X SSC 42.degree. C.; 1X SSC, 50% formamide B DNA:DNA <50
T.sub.B*; 1X SSC T.sub.B*; 1X SSC C DNA:RNA >50 67.degree. C.;
1X SSC -or- 67.degree. C.; 0.3X SSC 45.degree. C.; 1X SSC, 50%
formamide D DNA:RNA <50 T.sub.D*; 1X SSC T.sub.D*; 1X SSC E
RNA:RNA >50 70.degree. C.; 1X SSC -or- 70.degree. C.; 0.3X SSC
50.degree. C.; 1X SSC, 50% formamide F RNA:RNA <50 T.sub.F*; 1X
SSC T.sub.F*; 1X SSC G DNA:DNA >50 65.degree. C.; 4X SSC -or-
65.degree. C.; 1X SSC 42.degree. C.; 4X SSC, 50% formamide H
DNA:DNA <50 T.sub.H*; 4X SSC T.sub.H*; 4X SSC I DNA:RNA >50
67.degree. C.; 4X SSC -or- 67.degree. C.; 1X SSC 45.degree. C.; 4X
SSC, 50% formamide J DNA:RNA <50 T.sub.J*; 4X SSC T.sub.J; 4X
SSC K RNA:RNA >50 70.degree. C.; 4X SSC -or- 67.degree. C.; 1X
SSC 50.degree. C.; 4X SSC, 50% formamide L RNA:RNA <50 T.sub.L*;
2X SSC T.sub.L*; 2X SSC M DNA:DNA >50 50.degree. C.; 4X SSC -or-
50.degree. C.; 2X SSC 40.degree. C.; 6X SSC, 50% formamide N
DNA:DNA <50 T.sub.N*; 6X SSC T.sub.N*; 6X SSC O DNA:RNA >50
55.degree. C.; 4X SSC -or- 55.degree. C.; 2X SSC 42.degree. C.; 6X
SSC, 50% formamide P DNA:RNA <50 T.sub.P*; 6X SSC T.sub.P*; 6X
SSC Q RNA:RNA >50 60.degree. C.; 4X SSC -or- 60.degree. C.; 2X
SSC 45.degree. C.; 6X SSC, 50% formamide R RNA:RNA <50 T.sub.R*;
4X SSC T.sub.R*; 4X SSC .sup.1The hybrid length is that anticipated
for the hybridized region(s) of the hybridizing polynucleotides.
When hybridizing a polynucleotide to a target polynucleotide of
unknown sequence, the hybrid length is assumed to be that of the
hybridizing polynucleotide. When polynucleotides of known sequence
are hybridized, the hybrid length can be determined by aligning the
sequences of the polynucleotides and identifying the region or
regions of optimal sequence complementarity. .sup.2SSPE (1xSSPE is
0.15M NaCl, 10 mM NaH.sub.2PO.sub.4, and 1.25 mM EDTA, pH 7.4) can
be substituted for SSC (1xSSC is 0.15M NaCl and 15 mM sodium
citrate) in the hybridization and wash buffers; washes are
performed for 15 minutes after hybridization is complete. T.sub.B*
- T.sub.R*: The hybridization temperature for hybrids anticipated
to be less than 50 base pairs in length should be 5-10.degree. C.
less than the melting temperature (T.sub.m) of the hybrid, where Tm
is determined according to the following equations. For hybrids
less than 18 base pairs in length, T.sub.m(.degree. C.) = 2(# of A
+ T bases) + 4(# of G + C bases). For hybrids between 18 and 49
base pairs in length, T.sub.m(.degree. C.) = 81.5 +
16.6(log.sub.10Na.sup.+) + 0.41(% G + C) - (600/N), where N is the
number of bases in the hybrid, and Na.sup.+ is the concentration of
sodium ions in the hybridization buffer (Na.sup.+ for 1X SSC =
0.165M). Additional examples of stringency conditions for
polynucleotide hybridization are provided in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Chs. 9 & 11, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and
Ausubel et al., eds., Current Protocols in Molecular Biology,
Sects. 2.10 & 6.3-6.4, John Wiley & Sons, Inc. (1995),
herein incorporated by reference.
The isolated polynucleotides of the present invention may be used
as hybridization probes and primers to identify and isolate DNAs
having sequences encoding allelic variants of the disclosed
polynucleotides. Allelic variants are naturally occurring
alternative forms of the disclosed polynucleotides that encode
polypeptides that are identical to or have significant similarity
to the polypeptides encoded by the disclosed polynucleotides.
Preferably, allelic variants have at least 90% sequence identity
(more preferably, at least 95% identity; most preferably, at least
99% identity) with the disclosed polynucleotides.
The isolated polynucleotides of the present invention may also be
used as hybridization probes and primers to identify and isolate
DNAs having sequences encoding polypeptides homologous to the
disclosed polynucleotides. These homologs are polynucleotides and
polypeptides isolated from a different species than that of the
disclosed polypeptides and polynucleotides, or within the same
species, but with significant sequence similarity to the disclosed
polynucleotides and polypeptides. Preferably, polynucleotide
homologs have at least 50% sequence identity (more preferably, at
least 75% identity; most preferably, at least 90% identity) with
the disclosed polynucleotides, whereas polypeptide homologs have at
least 30% sequence identity (more preferably, at least 45%
identity; most preferably, at least 60% identity) with the
disclosed antibodies/polypeptides. Preferably, homologs of the
disclosed polynucleotides and polypeptides are those isolated from
mammalian species.
The isolated polynucleotides of the present invention may also be
used as hybridization probes and primers to identify cells and
tissues that express the antibodies of the present invention and
the conditions under which they are expressed.
Additionally, the isolated polynucleotides of the present invention
may be used to alter (i.e., enhance, reduce, or modify) the
expression of the genes corresponding to the polynucleotides of the
present invention in a cell or organism. These "corresponding
genes" are the genomic DNA sequences of the present invention that
are transcribed to produce the mRNAs from which the polynucleotides
of the present invention are derived.
The present invention also provides constructs in the form of
plasmids, vectors, transcription or expression cassettes which
comprise at least one nucleic acid of the invention as above.
The isolated polynucleotides of the present invention may be
operably linked to an expression control sequence for recombinant
production of the polypeptides of the present invention.
Additionally one of skill in the art will recognize that the
polynucleotides of the invention may be operably linked to
well-known nucleotide sequences encoding the constant region for
various antibody isotypes. For example, a polynucleotide of the
invention that encodes a light chain variable region(s) of the
invention (e.g., the sequence set forth in SEQ ID NOs:4 or 8) may
be operably linked to a nucleotide sequence that encodes the
constant region (or derivatives thereof) of either a .kappa. light
chain or .lamda. light chain, such that the expression of the
linked nucleotides will result in a full kappa or lambda light
chain with a variable region that specifically binds to and
neutralizes GDF-8. Similarly, a polynucleotide of the invention
that encodes a heavy chain variable region of the invention (e.g.,
the sequence set forth in SEQ ID NOs:2 or 6) may be operably linked
to a nucleotide sequence that encodes the constant region of a
heavy chain isotype (or derivatives thereof), e.g., IgM, IgD, IgE,
IgG and IgA. General methods of expressing recombinant proteins are
well known in the art. Such recombinant proteins may be expressed
in soluble form for use in treatment of disorders related to GDF-8
activity, e.g., muscle and bone degenerative disorders.
The recombinant expression vectors of the invention may carry
additional sequences, such as sequences that regulate replication
of the vector in host cells (e.g., origins of replication), tag
sequences such as histidine, and selectable marker genes. The
selectable marker gene facilitates selection of host cells into
which the vector has been introduced. For example, typically the
selectable marker gene confers resistance to drugs, such as G418,
hygromycin or methotrexate, on a host cell into which the vector
has been introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr.sup.- host
cells with methotrexate selection/amplification) and the neo gene
(for G418 selection).
Suitable vectors, containing appropriate regulatory sequences,
including promoter sequences, terminator sequences, polyadenylation
sequences, enhancer sequences, marker genes and other sequences as
appropriate, may be either chosen or constructed. Vectors may be
plasmids or viral, e.g., phage, or phagemid, as appropriate. For
further details see, for example, Molecular Cloning: a Laboratory
Manual: 2nd ed., Sambrook et al., Cold Spring Harbor Laboratory
Press, 1989. Many known techniques and protocols for manipulation
of nucleic acid, for example, in preparation of nucleic acid
constructs, mutagenesis, sequencing, introduction of DNA into cells
and gene expression, and analysis of proteins, are described in
detail in Current Protocols in Molecular Biology, 2nd ed., Ausubel
et al. eds., John Wiley & Sons, 1992.
The present invention also provides a host cell that comprises one
or more constructs as above. A nucleic acid encoding any CDR(H1,
H2, H3, L1, L2, or L3), VH or VL domain, or antigen-binding
fragment as provided herein, forms an aspect of the present
invention.
The present invention also includes a method of producing a peptide
by expressing the protein from the encoding nucleic acid in a host
cell. Expression may be achieved by culturing recombinant host
cells containing the nucleic acid under appropriate conditions.
Specific antibody fragments, VH and/or VL domains, and encoding
nucleic acid molecules and vectors according to the present
invention may be provided isolated and purified, e.g., from their
natural environment, in substantially pure or homogeneous form, or,
in the case of nucleic acids, free or substantially free of nucleic
acids or genes of origin other than the sequence encoding a
polypeptide with the required function.
A number of cell lines are suitable host cells for recombinant
expression of the polypeptides and antibodies of the present
invention. Mammalian host cell lines include, for example, COS
cells, CHO cells, 293T cells, A431 cells, 3T3 cells, CV-1 cells,
HeLa cells, L cells, BHK21 cells, HL-60 cells, U937 cells, HaK
cells, Jurkat cells, as well as cell strains derived from in vitro
culture of primary tissue and primary explants. Such host cells
also allow splicing of the polynucleotides of the invention that
consist of genomic DNA.
Alternatively, it may be possible to recombinantly produce the
polypeptides and antibodies of the present invention in lower
eukaryotes such as yeast or in prokaryotes. Potentially suitable
yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces
pombe, Kluyveromyces strains, and Candida strains. Potentially
suitable bacterial strains include Escherichia coli, Bacillus
subtilis, and Salmonella typhimurium. If the polypeptides of the
present invention are made in yeast or bacteria, it may be
necessary to modify them by, for example, phosphorylation or
glycosylation of appropriate sites, in order to obtain functional
proteins. Such covalent attachments may be accomplished using
well-known chemical or enzymatic methods.
The polypeptides and antibodies of the present invention may also
be recombinantly produced by operably linking the isolated
polynucleotides of the present invention to suitable control
sequences in one or more insect expression vectors, such as
baculovirus vectors, and employing an insect cell expression
system. Materials and methods for baculovirus/Sf9 expression
systems are commercially available in kit form (e.g., the
MAXBAC.RTM. kit, Invitrogen, Carlsbad, Calif.).
Following recombinant expression in the appropriate host cells, the
polypeptides and antibodies of the present invention may be
purified from culture medium or cell extracts using known
purification processes, such as gel filtration and ion exchange
chromatography. Purification may also include affinity
chromatography with agents known to bind the polypeptides and
antibodies of the present invention. These purification processes
may also be used to purify the polypeptides and antibodies of the
present invention from natural sources.
Alternatively, the polypeptides and antibodies of the present
invention may be recombinantly expressed in a form that facilitates
purification. For example, the polypeptides may be expressed as
fusions with proteins such as maltose-binding protein (MBP),
glutathione-S-transferase (GST), or thioredoxin (TRx). Kits for
expression and purification of such fusion proteins are
commercially available from New England BioLabs (Beverly, Mass.),
Pharmacia (Piscataway, N.J.), and Invitrogen, respectively. The
polypeptides and antibodies of the present invention can also be
tagged with a small epitope and subsequently identified or purified
using a specific antibody to the epitope. A preferred epitope is
the FLAG epitope, which is commercially available from Eastman
Kodak (New Haven, Conn.).
The polypeptides and antibodies of the present invention may also
be produced by known conventional chemical synthesis. Methods for
chemically synthesizing the polypeptides and antibodies of the
present invention are well known to those skilled in the art. Such
chemically synthetic polypeptides and antibodies may possess
biological properties in common with the natural purified
polypeptides and antibodies, and thus may be employed as
biologically active or immunological substitutes for the natural
polypeptides and antibodies.
A further aspect of the present invention provides a host cell
comprising nucleic acids, polypeptides, vectors, or antibodies and
fragments thereof as disclosed herein. A still further aspect
provides a method comprising introducing a nucleic acid of the
invention into a host cell. The introduction may employ any
available technique. For eukaryotic cells, suitable techniques may
include calcium phosphate transfection, DEAE-Dextran,
electroporation, liposome-mediated transfection and transduction
using a retrovirus or another virus, e.g., vaccinia or, for insect
cells, baculovirus. For bacterial cells, suitable techniques may
include calcium chloride transformation, electroporation and
infection using bacteriophage.
The introduction of nucleic acids may be followed by causing or
allowing protein production from the nucleic acid, e.g., by
culturing the host cells under conditions suitable for gene
expression. Such conditions are well known in the art.
III. Methods of Treating, Ameliorating, Preventing, and Inhibiting
the Progress of Bone, Adipose, Glucose Metabolism, Insulin and
Muscle Disorders
The involvement of GDF-8 in ALS, and the discovery of the novel
antibodies of the invention, enables methods for treating,
alleviating, and ameliorating GDF-8-associated disorders, e.g.,
muscle disorders, neuromuscular disorders, bone degenerative
disorders, metabolic or induced bone disorders, glucose metabolism
disorders, adipose disorders, and insulin-related disorders. In
addition, the antibodies allow for diagnosing, prognosing and
monitoring the progress of bone, muscle, adipose or insulin
disorders by measuring the level of GDF-8 in a biological sample.
In particular, the antibodies of the invention can be used to treat
an individual with ALS or other muscle disorder, or in a method of
distinguishing whether a patient is suffering from ALS or another
muscle disorder.
The antibodies and other molecules of the present invention are
useful to prevent, diagnose, or treat various medical disorders in
humans or animals. The antibodies can be used to inhibit or reduce
one or more activities associated with GDF-8. Most preferably, the
antibodies inhibit or reduce one or more of the activities of GDF-8
relative to unbound GDF-8 activities. In certain embodiments, the
activity of GDF-8, when bound by one or more anti-GDF-8 antibody is
inhibited at least 50%, preferably at least 60, 62, 64, 66, 68, 70,
72, 72, 76, 78, 80, 82, 84, 86, or 88%, more preferably at least
90, 91, 92, 93, or 94%, and even more preferably at least 95% to
100% relative to a mature GDF-8 protein that is not bound by one or
more of the anti-GDF-8 antibodies Inhibition or neutralization of
GDF-8 activity can be measured, e.g., in pGL3(CAGA).sub.12 reporter
gene assays (RGA) as described in Thies et al., supra, and in
ActRIIB receptor assays as illustrated in the Examples.
The medical disorders diagnosed, prognosed, monitored, treated,
ameliorated or prevented by GDF-8 antibodies are GDF-8-associated
disorders, e.g., muscle or neuromuscular disorders including, e.g.,
muscular dystrophy (MD; including Duchenne's muscular dystrophy),
amyotrophic lateral sclerosis (ALS), muscle atrophy, organ atrophy,
frailty, carpal tunnel syndrome, congestive obstructive pulmonary
disease, sarcopenia, cachexia, and other muscle wasting syndromes
(e.g., caused by other diseases and conditions). In addition, other
medical disorders that may be diagnosed, prognosed, monitored,
treated, ameliorated or prevented by the GDF-8 antibodies are
adipose tissue disorders such as obesity, type 2 diabetes, impaired
glucose tolerance, metabolic syndromes (e.g., syndrome X), insulin
resistance induced by trauma (such as burns or nitrogen imbalance),
or bone degenerative diseases (e.g., osteoarthritis, osteoporosis,
etc.). In preferred embodiments, the disorders that are diagnosed,
prognosed, monitored, treated, ameliorated or prevented by GDF-8
antibodies are muscular or neuromuscular disorders. In a more
preferred embodiment, the muscular or neuromuscular disorder that
is diagnosed, prognosed, monitored, treated, ameliorated or
prevented by anti-GDF-8 antibodies is either MD or ALS. In the most
preferred embodiment of the invention, the muscular or
neuromuscular disorder that is diagnosed, prognosed, monitored,
treated, ameliorated or prevented by GDF-8 antagonists of the
present invention, e.g., antibodies that inhibit GDF-8 activity, is
ALS.
Other medical disorders that may be diagnosed, prognosed,
monitored, treated, ameliorated or prevented by GDF-8 antagonists
are those associated with a loss of bone, which include
osteoporosis, especially in the elderly and/or postmenopausal
women, glucocorticoid-induced osteoporosis, osteopenia,
osteoarthritis, and osteoporosis-related fractures. Other target
metabolic bone diseases and disorders include low bone mass due to
chronic glucocorticoid therapy, premature gonadal failure, androgen
suppression, vitamin D deficiency, secondary hyperparathyroidism,
nutritional deficiencies, and anorexia nervosa. The antibodies are
preferably used to diagnose, prognose, monitor, treat, ameliorate
or prevent such disorders in mammals, particularly in humans.
The antibodies of the present invention are administered in
therapeutically effective amounts. Generally, a therapeutically
effective amount may vary with the subject's age, condition, and
sex, as well as the severity of the medical condition in the
subject. The dosage may be determined by a physician and adjusted,
as necessary, to suit observed effects of the treatment. Toxicity
and therapeutic efficacy of such compounds can be determined by
standard pharmaceutical procedures in cell cultures or experimental
animals, e.g., for determining the LD.sub.50 (the dose lethal to
50% of a population) and the ED.sub.50 (the dose therapeutically
effective in 50% of a population). The dose ratio between toxic and
therapeutic effects, i.e., the LD.sub.50/ED.sub.50, is the
therapeutic index, and antibodies exhibiting large therapeutic
indices are preferred.
The data obtained from cell culture assays and animal studies can
be used in formulating a range of dosage for use in humans. The
dosage of such compounds lies preferably within a range of
circulating concentrations that includes the ED.sub.50 with little
or no toxicity. The dosage may vary within this range depending
upon the form of dosage and the route of administration. For any
antibody used in the present invention, the therapeutically
effective dose can be estimated initially from cell culture assays.
A dose may be formulated in animal models to achieve a circulating
plasma concentration range that includes the IC.sub.50 (e.g., the
concentration of the test antibody which achieves a half-maximal
inhibition of symptoms or half-maximal inhibition of inhibition of
biological activity) as determined in cell culture. Levels in
plasma may be measured, for example, by high performance liquid
chromatography. The effects of any particular dosage can be
monitored by a suitable bioassay. Examples of suitable bioassays
include, but are not limited to, DNA replication assays,
transcription-based assays, GDF-8 protein/receptor binding assays,
creatine kinase assays, assays based on the differentiation of
preadipocytes, assays based on glucose uptake in adipocytes, and
immunological assays.
Generally, the compositions are administered so that antibodies or
their binding fragments are given at a dose from 1 .mu.g/kg to 150
mg/kg, 1 .mu.g/kg to 100 mg/kg, 1 .mu.g/kg to 50 mg/kg, 1 .mu.g/kg
to 20 mg/kg, 1 .mu.g/kg to 10 mg/kg, 1 .mu.g/kg to 1 mg/kg, 10
.mu.g/kg to 1 mg/kg, 10 .mu.g/kg to 100 .mu.g/kg, 100 .mu.g to 1
mg/kg, and 500 .mu.g/kg to 1 mg/kg. Preferably, the antibodies are
given as a bolus dose to maximize the circulating levels of
antibodies for the greatest length of time after the dose.
Continuous infusion may also be used before, after, or in place of
the bolus dose.
IV. Methods of Identifying Therapeutic Agents
Yet another aspect of the invention provides a method of
identifying therapeutic agents useful in treatment of muscle, e.g.,
glucose metabolism, adipose, and bone disorders. Appropriate
screening assays, e.g., ELISA-based assays, are known in the art.
In such a screening assay, a first binding mixture is formed by
combining an antibody of the invention and its ligand, GDF-8, and
the amount of binding between the ligand and the antibody in the
first binding mixture (M.sub.0) is measured. A second binding
mixture is also formed by combining the antibody, the ligand, and a
compound or agent to be screened, and the amount of binding between
the ligand and the antibody in the second binding mixture (M.sub.1)
is measured. The amounts of binding in the first and second binding
mixtures are then compared, for example, by calculating the
M.sub.1/M.sub.0 ratio. The compound or agent is considered to be
capable of inhibiting GDF-8 activity if a decrease in binding in
the second binding mixture as compared to the first binding mixture
is observed (i.e., M.sub.1/M.sub.0<1). The formulation and
optimization of binding mixtures is within the level of skill in
the art; such binding mixtures may also contain buffers and salts
necessary to enhance or to optimize binding, and additional control
assays may be included in the screening assay of the invention.
Compounds found to reduce the antibody-ligand binding by at least
about 10% (i.e., M.sub.1/M.sub.0<0.9), preferably greater than
about 30%, may thus be identified and then, if desired, secondarily
screened for the capacity to inhibit GDF-8 activity in other assays
such as the ActRIIB binding assay, or other cell-based and in vivo
assays as described in the Examples or well known in the art.
V. Small Molecules
Inhibiting GDF-8 activity in an organism (or subject) afflicted
with (or at risk for) a GDF-8-associated disorder, or in a cell
from such an organism involved in such disorders, may also be
achieved through the use of antagonist small molecules (usually
organic small molecules) that antagonize, i.e., inhibit the
activity of, GDF-8. Novel antagonistic small molecules may be
identified by the screening methods described above and may be used
in the treatment methods of the present invention described
herein.
Conversely, increasing GDF-8 activity in an organism (or subject)
afflicted with (or at risk for) a disorder related to decreased
GDF-8 expression and/or activity or a disorder related to decreased
GDF-8 levels may also be achieved through the use of small
molecules (usually organic small molecules) that agonize, i.e.,
enhance the activity of, GDF-8. Novel agonistic small molecules may
be identified by the screening methods described above and may be
used in the treatment methods of the present invention described
herein.
VI. Methods of Diagnosing, Prognosing, and Monitoring the Progress
of Bone, Adipose, Glucose Metabolism, and Muscle Disorders
In addition to treating, e.g., muscle, bone, glucose metabolism,
and adipose disorders, the present invention provides methods for
diagnosing such disorders by detecting the decrease or increase of
GDF-8 in a biological sample, e.g., serum, plasma, bronchoalveolar
lavage fluid, sputum, biopsies (e.g., of muscle), etc. "Diagnostic"
or "diagnosing" means identifying the presence or absence of a
pathologic condition. Diagnostic methods involve detecting the
presence of GDF-8 by, e.g., determining a test amount of GDF-8
polypeptide in a biological sample from a subject (human or
nonhuman mammal), and comparing the test amount with a normal
amount or range (e.g., an amount or range from an individual(s)
known not to suffer from such a disorder) for the GDF-8
polypeptide. While a particular diagnostic method may not provide a
definitive diagnosis of ALS or other GDF-8-associated disorders, it
suffices if the method provides a positive indication that aids in
diagnosis.
The present invention also provides methods for prognosing ALS or
other muscle disorders, or e.g., bone, glucose metabolism, and
adipose disorders by detecting upregulation of GDF-8. "Prognostic"
or "prognosing" means predicting the probable development and/or
severity of a pathologic condition. Prognostic methods involve
determining the test amount of GDF-8 in a biological sample from a
subject, and comparing the test amount to a prognostic amount or
range (e.g., an amount or range from individuals with varying
severities of, e.g., ALS) for GDF-8. Various amounts of the GDF-8
in a test sample are consistent with certain prognoses for ALS or
other GDF-8-associated disorders. The detection of an amount of
GDF-8 at a particular prognostic level provides a prognosis for the
subject.
The present invention also provides methods for monitoring the
course of ALS or other GDF-8-associated disorders by detecting the
upregulation or downregulation of GDF-8. Monitoring methods involve
determining the test amounts of GDF-8 in biological samples taken
from a subject at a first and second time, and comparing the
amounts. A change in amount of GDF-8 between the first and second
time indicates a change in the course of, e.g., severity of, ALS or
other GDF-8-associated disorders. A skilled artisan will recognize
that in GDF-8-associated disorders similar to ALS, e.g., where an
increase in muscle mass is desirable, a decrease in amount of GDF-8
and/or GDF-8 activity between the first and second time indicates
remission of the disorder, and an increase in amount indicates
progression of the disorder. Such monitoring assays are also useful
for evaluating the efficacy of a particular therapeutic
intervention (e.g., disease attenuation and/or reversal) in
patients being treated for ALS or other GDF-8-associated
disorders.
The antibodies of the present invention may be used for diagnosis,
prognosis or monitoring by detecting the presence of GDF-8 in vivo
or in vitro. Such detection methods are well known in the art and
include ELISA, radioimmunoassay, immunoblot, Western blot,
immunofluorescence, immunoprecipitation, and other comparable
techniques. The antibodies may further be provided in a diagnostic
kit that incorporates one or more of these techniques to detect
GDF-8. Such a kit may contain other components, packaging,
instructions, or other material to aid the detection of the protein
and use of the kit.
Where the antibodies are intended for diagnostic, prognostic, or
monitoring purposes, it may be desirable to modify them, for
example, with a ligand group (such as biotin) or a detectable
marker group (such as a fluorescent group, a radioisotope or an
enzyme). If desired, the antibodies (whether polyclonal or
monoclonal) may be labeled using conventional techniques. Suitable
labels include fluorophores, chromophores, radioactive atoms,
electron-dense reagents, enzymes, and ligands having specific
binding partners. Enzymes are typically detected by their activity.
For example, horseradish peroxidase can be detected by its ability
to convert tetramethylbenzidine (TMB) to a blue pigment,
quantifiable with a spectrophotometer. Other suitable labels may
include biotin and avidin or streptavidin, IgG and protein A, and
the numerous receptor-ligand couples known in the art. Other
permutations and possibilities will be readily apparent to those of
ordinary skill in the art, and are considered as equivalents within
the scope of the instant invention.
VII. Pharmaceutical Compositions and Methods of Administration
The present invention provides compositions comprising a GDF-8
antagonist of the invention, i.e., polypeptides, polynucleotides,
vectors, antibodies, antibody fragments, and small molecules. Such
compositions may be suitable for pharmaceutical use and
administration to patients. The compositions typically comprise one
or more molecules of the present invention, preferably an antibody,
and a pharmaceutically acceptable excipient. The anti-GDF-8
antibodies of the present invention can be used in vitro, ex vivo,
or incorporated into a pharmaceutical composition when combined
with a pharmaceutically acceptable carrier. As used herein, the
phrase "pharmaceutically acceptable excipient" includes any and all
solvents, solutions, buffers, dispersion medias, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like, that are compatible with
pharmaceutical administration. Such a composition may contain, in
addition to the antibodies of the invention and carrier, various
diluents, fillers, salts, buffers, stabilizers, solubilizers, and
other materials well known in the art. The term "pharmaceutically
acceptable" means a nontoxic material that does not interfere with
the effectiveness of the biological activity of the active
ingredient(s). The characteristics of the carrier will depend on
the route of administration. The use of such media and agents for
pharmaceutically active substances is well known in the art. The
compositions may also contain other active compounds providing
supplemental, additional, or enhanced therapeutic functions. The
pharmaceutical compositions may also be included in a container,
pack, or dispenser together with instructions for
administration.
The pharmaceutical composition of the invention may be in the form
of a liposome in which an antibody of the invention is combined, in
addition to other pharmaceutically acceptable carriers, with
amphipathic agents such as lipids that exist in aggregated form as
micelles, insoluble monolayers, liquid crystals, or lamellar layers
while in aqueous solution. Suitable lipids for liposomal
formulation include, without limitation, monoglycerides,
diglycerides, sulfatides, lysolecithin, phospholipids, saponin,
bile acids, and the like. Preparation of such liposomal
formulations is within the level of skill in the art.
As used herein, the term "therapeutically effective amount" means
the total amount of each active component of the pharmaceutical
composition or method that is sufficient to show a meaningful
patient benefit, e.g., amelioration of symptoms of, healing of, or
increase in rate of healing of such conditions. When applied to an
individual active ingredient, administered alone, the term refers
to that ingredient alone. When applied to a combination, the term
refers to combined amounts of the active ingredients that result in
the therapeutic effect, whether administered in combination,
serially or simultaneously.
In practicing the method of treatment or use of the present
invention, a therapeutically effective amount of, e.g., an antibody
that binds to GDF-8 and interferes with GDF-8 signaling is
administered to a subject, e.g., mammal (e.g., a human). An
antibody of the invention may be administered in accordance with
the method of the invention either alone or in combination with
other therapies such as anti-inflammatory agents. When
coadministered with one or more agents, an antibody of the
invention may be administered either simultaneously with the second
agent, or sequentially. If administered sequentially, the attending
physician will decide on the appropriate sequence of administering
an antibody of the invention in combination with other agents.
In one embodiment, the antibodies of the invention, e.g.,
pharmaceutical compositions thereof, are administered in
combination therapy, i.e., combined with other agents, e.g.,
therapeutic agents, that are useful for treating pathological
conditions or disorders, such as muscle disorders, neuromuscular
disorders, bone degenerative disorders, metabolic or induced bone
disorders, adipose disorders, glucose metabolism disorders or
insulin-related disorders, e.g., as well as allergic and
inflammatory disorders. The term "in combination" in this context
means that the agents are given substantially contemporaneously,
either simultaneously or sequentially. If given sequentially, at
the onset of administration of the second compound, the first of
the two compounds is preferably still detectable at effective
concentrations at the site of treatment or in the subject.
A pharmaceutical composition of the invention is formulated to be
compatible with its intended route of administration. Methods to
accomplish the administration are known to those of ordinary skill
in the art. It may also be possible to obtain compositions which
may be topically or orally administered, or which may be capable of
transmission across mucous membranes. Administration of an antibody
of the invention used in a pharmaceutical composition to practice
the method of the present invention can be carried out in a variety
of conventional ways, such as oral ingestion, inhalation,
cutaneous, subcutaneous, or intravenous injection.
Solutions or suspensions used for intradermal or subcutaneous
application typically include one or more of the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates; and agents for the adjustment of tonicity such as
sodium chloride or dextrose. The pH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide. Such
preparations may be enclosed in ampoules, disposable syringes or
multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injection include sterile
aqueous solutions or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or
dispersion. For intravenous administration, suitable carriers
include physiological saline, bacteriostatic water, CREMOPHOR.TM.
EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In
all cases, the composition must be sterile and should be fluid to
the extent that easy syringability exists. It must be stable under
the conditions of manufacture and storage and must be preserved
against the contaminating action of microorganisms such as bacteria
and fungi. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. Prevention of the action
of microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, and sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
When a therapeutically effective amount of an antibody of the
invention is administered by, e.g., intravenous, cutaneous or
subcutaneous injection, the binding agent will be in the form of a
pyrogen-free, parenterally acceptable aqueous solution. The
preparation of such parenterally acceptable protein solutions,
having due regard to pH, isotonicity, stability, and the like, is
within the skill in the art. A preferred pharmaceutical composition
for intravenous, cutaneous, or subcutaneous injection should
contain, in addition to binding agents, an isotonic vehicle such as
sodium chloride injection, Ringer's injection, dextrose injection,
dextrose and sodium chloride injection, lactated Ringer's
injection, or other vehicle as known in the art. The pharmaceutical
composition(s) of the present invention may also contain
stabilizers, preservatives, buffers, antioxidants, or other
additive known to those of skill in the art.
The amount of an antibody of the invention (or other antagonist of
the invention) in the pharmaceutical composition of the present
invention will depend upon the nature and severity of the condition
being treated, and on the nature of prior treatments undergone by
the patient. Ultimately, the attending physician will decide the
amount of antibody with which to treat each individual patient.
Initially, an attending physician administers low doses of antibody
and observes the patient's response. Larger doses of antibody may
be administered until the optimal therapeutic effect is obtained
for the patient, and at that point the dosage is generally not
increased further. It is contemplated that the various
pharmaceutical compositions used to practice the method of the
present invention should contain about 0.1 .mu.g to 50 mg antibody
per kg body weight.
The duration of therapy using the pharmaceutical composition of the
present invention will vary, depending on the severity of the
disease being treated and the condition and potential idiosyncratic
response of each individual patient. It is contemplated that the
duration of each application of antibody will be via, e.g., the
subcutaneous route and, e.g., in the range of once a week.
Ultimately the attending physician will decide on the appropriate
duration of therapy using the pharmaceutical composition of the
present invention.
Oral compositions generally include an inert diluent or an edible
carrier. They can be enclosed in gelatin capsules or compressed
into tablets. For the purpose of oral therapeutic administration,
the GDF-8 antagonist (e.g., antibody, small molecule, etc.) can be
incorporated with excipients and used in the form of tablets or
capsules. Pharmaceutically compatible binding agents, and/or
adjuvant materials can be included as part of the composition. The
tablets, pills, capsules, and the like can contain any of the
following ingredients, or compounds of a similar nature; a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose; a disintegrating agent such as
alginic acid, PRIMOGEL.TM., or corn starch; a lubricant such as
magnesium stearate or STEROTES.TM., a glidant such as colloidal
silicon dioxide; a sweetening agent such as sucrose or saccharin;
or a flavoring agent such as peppermint, methyl salicylate, or
orange flavoring.
When a therapeutically effective amount of a pharmaceutical
composition of the invention, e.g., an antibody that binds to GDF-8
and interferes with GDF-8 signaling, is administered orally, the
binding agent will be in the form of a tablet, capsule, powder,
solution or elixir. When administered in tablet form, the
pharmaceutical composition of the invention may additionally
contain a solid carrier such as a gelatin or an adjuvant. The
tablet, capsule, and powder contain from about 5 to 95% binding
agent, and preferably from about 25 to 90% binding agent. When
administered in liquid form, a liquid carrier such as water,
petroleum, oils of animal or plant origin such as peanut oil,
mineral oil, soybean oil, or sesame oil, or synthetic oils may be
added (after taking into account the allergies of the individual
patient and/or vast population of individuals to such liquid
carriers). The liquid form of the pharmaceutical composition may
further contain physiological saline solution, dextrose or other
saccharide solution, or glycols such as ethylene glycol, propylene
glycol or polyethylene glycol. When administered in liquid form,
the pharmaceutical composition contains from about 0.5 to 90% by
weight of the binding agent, and preferably from about 1 to 50% the
binding agent.
For administration by inhalation, a GDF-8 antagonist is delivered
in the form of an aerosol spray from a pressured container or
dispenser, which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer. Accordingly, the compounds
described herein can be administered by inhalation to pulmonary
tissue. The term "pulmonary tissue" as used herein refers to any
tissue of the respiratory tract and includes both the upper and
lower respiratory tract, except where otherwise indicated. One or
more GDF-8 antibodies can be administered in combination with one
or more of the existing modalities for treating pulmonary
diseases.
In one example, the compound is formulated for a nebulizer. In one
embodiment, the compound can be stored in a lyophilized form (e.g.,
at room temperature) and reconstituted in solution prior to
inhalation.
It is also possible to formulate the compound for inhalation using
a medical device, e.g., an inhaler (see, e.g., U.S. Pat. No.
6,102,035 (a powder inhaler) and U.S. Pat. No. 6,012,454 (a dry
powder inhaler)). The inhaler can include separate compartments for
the active compound at a pH suitable for storage and another
compartment for a neutralizing buffer, and a mechanism for
combining the compound with a neutralizing buffer immediately prior
to atomization. In one embodiment, the inhaler is a metered dose
inhaler.
Although not necessary, delivery enhancers such as surfactants can
be used to further enhance pulmonary delivery. A "surfactant" as
used herein refers to a compound having hydrophilic and lipophilic
moieties that promote absorption of a drug by interacting with an
interface between two immiscible phases. Surfactants are useful
with dry particles for several reasons, e.g., reduction of particle
agglomeration, reduction of macrophage phagocytosis, etc. When
coupled with lung surfactant, a more efficient absorption of the
compound can be achieved because surfactants, such as DPPC, will
greatly facilitate diffusion of the compound. Surfactants are well
known in the art and include, but are not limited to,
phosphoglycerides, e.g., phosphatidylcholines,
L-alpha-phosphatidylcholine dipalmitoyl (DPPC) and diphosphatidyl
glycerol (DPPG); hexadecanol; fatty acids; polyethylene glycol
(PEG); polyoxyethylene-9-; auryl ether; palmitic acid; oleic acid;
sorbitan trioleate (Span 85); glycocholate; surfactin; poloxomer;
sorbitan fatty acid ester; sorbitan trioleate; tyloxapol; and
phospholipids.
Systemic administration can also be by transmucosal or transdermal
means. For example, in the case of antibodies that comprise the Fc
portion, compositions may be capable of transmission across mucous
membranes (e.g., intestine, mouth, or lungs) via the FcRn
receptor-mediated pathway (e.g., U.S. Pat. No. 6,030,613). In
general, transmucosal administration can be accomplished, for
example, through the use of lozenges, nasal sprays, inhalers, or
suppositories. For transdermal administration, the active compounds
are formulated into ointments, salves, gels, patches or creams as
generally known in the art. For transmucosal or transdermal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
generally known in the art, and include, for example, detergents,
bile salts, and fusidic acid derivatives.
Pharmaceutical compositions may also consist of compositions
suitable for gene therapy, i.e., compositions comprised of the
polynucleotides disclosed herein. In the case of gene therapy, the
pharmaceutically acceptable carrier may include, e.g., lipids,
collagen spheres, cationic emulsion systems, water, saline buffers,
viral vectors, chylomicron remnants, polymer nanoparticles (e.g.,
gelatin-DNA or chitosan-DNA), gold particles, polymer complexes,
lipoplexes, polyplexes, etc. (see, e.g., Gardlik et al. (2005) Med.
Sci. Monit. 11(4):RA110-21).
Stabilization and Retention
In one embodiment, a GDF-8 antibody is physically associated with a
moiety that improves its stabilization and/or retention in
circulation, e.g., in blood, serum, lymph, bronchopulmonary or
bronchoalveolar lavage, or other tissues, e.g., by at least 1.5, 2,
5, 10, or 50 fold.
The antagonists of the invention may be prepared with carriers that
will protect against rapid elimination from the body, such as a
controlled-release formulation, including implants and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. Liposomal suspensions
containing a GDF-8 antagonist, e.g., one or more anti-GDF-8
antibodies, can also be used as pharmaceutically acceptable
carriers. These can be prepared according to methods known to those
skilled in the art.
For example, a GDF-8 antibody can be associated with a polymer,
e.g., a substantially nonantigenic polymer, such as polyalkylene
oxides or polyethylene oxides. Suitable polymers will vary
substantially by weight. Polymers having molecular number average
weights ranging from about 200 to about 35,000 (or about 1,000 to
about 15,000, or about 2,000 to about 12,500) can be used.
For example, a GDF-8 antibody can be conjugated to a water-soluble
polymer, e.g., hydrophilic polyvinyl polymers, e.g.,
polyvinylalcohol and polyvinylpyrrolidone. A nonlimiting list of
such polymers include polyalkylene oxide homopolymers such as
polyethylene glycol (PEG) or polypropylene glycols,
polyoxyethylenated polyols, copolymers thereof and block copolymers
thereof, provided that the water solubility of the block copolymers
is maintained. Additional useful polymers include polyoxyalkylenes
such as polyoxyethylene, polyoxypropylene, and block copolymers of
polyoxyethylene and polyoxypropylene (Pluronics);
polymethacrylates; carbomers; branched or unbranched
polysaccharides, which comprise the saccharide monomers D-mannose,
D- and L-galactose, fucose, fructose, D-xylose, L-arabinose,
D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic
acid (e.g., polymannuronic acid, or alginic acid), D-glucosamine,
D-galactosamine, D-glucose and neuraminic acid including
homopolysaccharides and heteropolysaccharides such as lactose,
amylopectin, starch, hydroxyethyl starch, amylose, dextrane
sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit
of acid mucopolysaccharides, e.g., hyaluronic acid; polymers of
sugar alcohols such as polysorbitol and polymannitol; heparin,
etc.
Other compounds can also be attached to the same polymer, e.g., a
cytotoxin, a label, or another targeting agent, e.g., another GDF-8
antibody or an unrelated ligand. Mono-activated, alkoxy-terminated
polyalkylene oxides (PAOs), e.g., monomethoxy-terminated
polyethylene glycols (mPEGs), C.sub.1-4 alkyl-terminated polymers,
and bis-activated polyethylene oxides (glycols) can be used for
cross-linking (see, e.g., U.S. Pat. No. 5,951,974).
In one embodiment, the polymer prior to cross-linking to the ligand
need not be, but preferably is, water-soluble. Generally, after
cross-linking, the product is water-soluble, e.g., exhibits a water
solubility of at least about 0.01 mg/ml, and more preferably at
least about 0.1 mg/ml, and still more preferably at least about 1
mg/ml. In addition, the polymer should not be highly immunogenic in
the conjugate form, nor should it possess viscosity that is
incompatible with intravenous infusion, aerosolization, or
injection, if the conjugate is intended to be administered by such
routes.
In one embodiment, the polymer contains only a single group that is
reactive. This helps to avoid cross-linking of ligand molecules to
one another. However, it is within the scope herein to maximize
reaction conditions to reduce cross-linking between ligand
molecules, or to purify the reaction products through gel
filtration or ion exchange chromatography to recover substantially
homogenous derivatives. In other embodiments, the polymer contains
two or more reactive groups for the purpose of linking multiple
ligands to the polymer backbone. Again, gel filtration or ion
exchange chromatography can be used to recover the desired
derivative in substantially homogeneous form.
The molecular weight of the polymer can range up to about 500,000
D, and preferably is at least about 20,000 D, or at least about
30,000 D, or at least about 40,000 D. The molecular weight chosen
can depend upon the effective size of the conjugate to be achieved,
the nature (e.g., structure, such as linear or branched) of the
polymer, and the degree of derivatization.
A covalent bond can be used to attach a GDF-8 antibody to a
polymer, for example, cross-linking to the N-terminal amino group
of the ligand and epsilon amino groups found on lysine residues of
the ligand, as well as other amino, imino, carboxyl, sulfhydryl,
hydroxyl or other hydrophilic groups. The polymer may be covalently
bonded directly to the GDF-8 antibody without the use of a
multifunctional (ordinarily Afunctional) cross-linking agent.
Covalent binding to amino groups is accomplished by known
chemistries based upon cyanuric chloride, carbonyl diimidazole,
aldehyde-reactive groups (PEG alkoxide plus diethyl acetyl of
bromoacetaldehyde; PEG plus DMSO and acetic anhydride, or PEG
chloride plus the phenoxide of 4-hydroxybenzaldehyde, activated
succinimidyl esters, activated dithiocarbonate PEG,
2,4,5-trichlorophenylcloroformate or P-nitrophenylchloroformate
activated PEG.) Carboxyl groups can be derivatized by coupling
PEG-amine using carbodiimide. Sulfhydryl groups can be derivatized
by coupling to maleimido-substituted PEG (e.g., alkoxy-PEG amine
plus sulfosuccinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (see WO 97/10847)
or PEG-maleimide). Alternatively, free amino groups on the ligand
(e.g., epsilon amino groups on lysine residues) can be thiolated
with 2-imino-thiolane (Traut's reagent) and then coupled to
maleimide-containing derivatives of PEG, e.g., as described in
Pedley et al. (1994) Br. J. Cancer 70:1126-30.
Functionalized PEG polymers that can be attached to a GDF-8
antibody are available, e.g., from Shearwater Polymers, Inc.
(Huntsville, Ala.). Such commercially available PEG derivatives
include, e.g., amino-PEG, PEG amino acid esters, PEG-hydrazide,
PEG-thiol, PEG-succinate, carboxymethylated PEG, PEG-propionic
acid, PEG amino acids, PEG succinimidyl succinate, PEG succinimidyl
propionate, succinimidyl ester of carboxymethylated PEG,
succinimidyl carbonate of PEG, succinimidyl esters of amino acid
PEGs, PEG-oxycarbonylimidazole, PEG-nitrophenyl carbonate, PEG
tresylate, PEG-glycidyl ether, PEG-aldehyde, PEG vinylsulfone,
PEG-maleimide, PEG-orthopyridyl-disulfide, heterofunctional PEGs,
PEG vinyl derivatives, PEG silanes, and PEG phospholides. The
reaction conditions for coupling these PEG derivatives may vary
depending on the GDF-8 antibody, the desired degree of PEGylation,
and the PEG derivative utilized. Some factors involved in the
choice of PEG derivatives include: the desired point of attachment
(such as lysine or cysteine R-groups), hydrolytic stability and
reactivity of the derivatives, stability, toxicity and antigenicity
of the linkage, suitability for analysis, etc. Specific
instructions for the use of any particular derivative are available
from the manufacturer.
The conjugates of a GDF-8 antibody and a polymer can be separated
from the unreacted starting materials, e.g., by gel filtration or
ion exchange chromatography, or other forms of chromatography,
e.g., HPLC. Heterologous species of the conjugates are purified
from one another in the same fashion. Resolution of different
species (e.g., containing one or two PEG residues) is also possible
due to the difference in the ionic properties of the unreacted
amino acids (see, e.g., WO 96/34015).
The polynucleotides and proteins of the present invention are
expected to exhibit one or more of the uses or biological
activities (including those associated with assays cited below)
identified herein. Uses or activities described for proteins of the
present invention may be provided by administration or use of such
proteins, or by administration or use of polynucleotides encoding
such proteins (such as, e.g., in gene therapies or vectors suitable
for introduction of DNA).
It may be advantageous to formulate oral or parenteral compositions
in dosage unit form for ease of administration and uniformity of
dosage. Dosage unit form as used herein refers to physically
discrete units suited as unitary dosages for the subject to be
treated, each unit containing a predetermined quantity of active
compound calculated to produce the desired therapeutic effect in
association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on the unique characteristics of
the active compound, the particular therapeutic effect to be
achieved, and the limitations inherent in the art of formulating
such an active compound for the treatment of individuals.
Another aspect of the present invention accordingly relates to kits
for carrying out the administration of the GDF-8 antibodies of the
invention, e.g., with or without other therapeutic compounds, or
for using the anti-GDF-8 antibodies as a research or therapeutic
tool to determine the presence and/or level of GDF-8 in a
biological sample, such as an ELISA kit. In one embodiment, the kit
comprises one or more anti-GDF-8 antibodies formulated in a
pharmaceutical carrier, and at least one agent, e.g., a therapeutic
agent, formulated as appropriate, in one or more separate
pharmaceutical preparations.
The Examples which follow are set forth to aid in the understanding
of the invention but are not intended to, and should not be
construed to, limit the scope of the invention in any way. The
Examples do not include detailed descriptions of conventional
methods, such as hybridoma formation, ELISA, proliferation assays,
flow cytometric analysis and recombinant DNA techniques. Such
methods are well known to those of ordinary skill in the art.
The entire contents of all references, patents, published patent
applications, and other patent documents cited throughout this
application are hereby incorporated by reference herein.
EXAMPLES
Example 1
Creation and Identification of Anti-GDF-8 Antibody RK35
Human GDF-8 protein (mature GDF-8 and GDF-8 propeptide) and BMP-11
protein were isolated and characterized as described in U.S.
Published Patent Application No. 2004/0142382.
Six female myostatin knockout BALB/c mice (8 weeks old; McPherron
et al., supra) were immunized by subcutaneous injections with 20
.mu.g of recombinant GDF-8 dimer in Freund's complete adjuvant.
Several booster injections of the same amount of antigen in
Freund's incomplete adjuvant were given at 2-week intervals and a
final intravenous injection (tail vein) of 2 .mu.g in PBS was given
prior to the fusion. Splenocytes from two of the mice demonstrating
the highest antibody titers were fused with mouse myeloma cells
(ATCC Accession No. P3X63.Ag8.653) using standard techniques (Oi
and Herzenberger (1980) In: Mishell, B. B., Shiigi, S. M., Henry,
C., Mishell, R. I. (Eds.), Selected Methods in Cellular Immunology
W.H. Freemen, San Francisco, pp. 351-72). After 10-14 days, the
supernatants were harvested and screened for anti-GDF-8 antibody
production by solid and solution phase ELISA (Whittemore et al.,
supra). Standard ELISA techniques and the pGL3-(CAGA).sub.12
reporter assay (Theis et al (2001) Growth Factors 18:251-59) were
used to determine the IC.sub.50 for inhibition of binding of
myostatin to its receptor, ActRIIB, using a chimeric ActRIIB-Fc
generated by fusing the extracellular domain of the human
ActRIIB-Fc receptor with human IgG1 Fc region. Hybridomas chosen
for further studies were rendered monoclonals by repeated limiting
dilution to ensure monoclonality. Monoclonal antibody RK35 was
selected for further study.
Example 2
RK35 Monoclonal Antibody has High Affinity for GDF-8 and Exhibits
Neutralization Activity
Example 2.1
Experimental Procedures
For the ELISA, biotinylated GDF-8 was coated overnight at 4.degree.
C. onto 96-well streptavidin microtiter plates (Pierce, Rockford,
Ill.) at 1 .mu.g/ml. After coating, the solutions were removed from
the wells, and the plates blocked for 1 hour at room temperature in
SuperBlock solution (Pierce). Plates were rinsed with PBS, and 100
.mu.l of RK35 antibody was added to the wells at various
concentrations. The plates were incubated at room temperature for 1
hour and then washed with PBS. To each well, 100 IA of a 1:5000
dilution of anti-huIgG-HRP conjugate (Southern Biotech, Birmingham,
Ala.) was added and the plates were incubated at room temperature
for 1 hour. Each plate was washed three times with PBS. TMB
substrate (100 .mu.l) was added to each well and incubated until
color development. The reaction was stopped by the addition of 100
.mu.l of 0.18 M H.sub.2SO.sub.4. The signal generated was measured
by reading the absorbance at 450 nm using a microtiter plate
reader. Binding to GDF-8 was confirmed using human isotype control
antibody.
Recombinant ActRIIB-Fc chimera (R&D Systems, Minneapolis,
Minn., Cat. No. 339-RB/CF) was coated on 96-well flat-bottom assay
plates (Costar, N.Y., Cat. No. 3590) at 1 .mu.g/ml in 0.2 M sodium
carbonate buffer overnight at 4.degree. C. Plates were then blocked
with 1 mg/ml bovine serum albumin and washed following standard
ELISA protocol. Aliquots (100 .mu.l) of biotinylated GDF-8 or
BMP-11 were added to the blocked ELISA plate at various
concentrations, incubated for 1 hr, washed, and the amount of bound
GDF-8 or BMP-11 was detected by streptavidin-horseradish peroxidase
(SA-HRP, BD PharMingen, San Diego, Calif., Cat. No. 13047E)
followed by the addition of TMB (KPL, Gaithersburg, Md., Cat. No.
50-76-04). Colorimetric measurements were taken at 450 nm in a
Molecular Devices microplate reader. To analyze the inhibitory
activity, RK35 was tested at various concentrations by
preincubation with 20 ng/ml GDF-8 or 20 ng/ml BMP-11. After
incubation for 1 hr at room temperature, 100 .mu.A of RK35 and
GDF-8 or BMP-11 mixture was added to the plate. Detection and
quantitation of bound factor is described in Whittemore et al.
(2003) Biochem. Biophys. Res. Commun. 300:965-71.
To demonstrate the activity of GDF-8, a reporter gene assay (RGA)
was developed using a reporter vector pGL3(CAGA).sub.12 expressing
luciferase under control of TGF-.beta. induced promoter. The CAGA
is a TGF-.beta.-responsive sequence within the promoter of the
TGF-.beta.-induced gene PAI-1 (Denner et al. (1998) EMBO J. 17:
3091-3100). A reporter vector containing 12 CAGA boxes was made
using the basic luciferase reporter plasmid pGL3 (Promega, Madison,
Wis.). The TATA box and transcription initiation site from the
adenovirus major later promoter (-35/+10) was inserted between the
BglII and HindIII sites. Oligonucleotides containing 12 repeats of
the CAGA boxes, i.e., AGCCAGACA, were annealed and cloned into the
XhoI site. The human rhabdomyosarcoma cell line A204 (ATCC HTB-82)
was transiently transfected with pGL3(CAGA).sub.12 using FuGENE 6
transfection reagent (Boehringer Manheim, Germany). Following
transfection, cells were cultured on 96-well plates in McCoy's 5A
medium supplemented with 2 mM glutamine, 100 U/ml streptomycin, 100
.mu.g/ml penicillin and 10% fetal calf serum for 16 hrs. Cells were
then treated with or without 10 ng/ml GDF-8 in McCoy's 5A medium
with glutamine, streptomycin, penicillin, and 1 mg/ml bovine serum
albumin for 6 hrs at 37.degree. C. Luciferase was quantified in the
treated cells using the Luciferase Assay System (Promega). To test
the inhibitory activity of RK35, GDF-8 was preincubated with the
antibody for 1 hr at room temperature. This mixture was then added
to the transfected cells and cells were incubated for 6 hrs at
37.degree. C. Luciferase was quantified using the Luciferase Assay
System (Promega).
Example 2.2
Results
A high affinity mouse monoclonal antibody to myostatin was
generated by immunizing GDF-8 knockout mice with purified
recombinant human GDF-8, which is identical in amino acid sequence
to mature murine myostatin (McPherron et al., 1997). The RK35
antibody bound with high affinity to GDF-8 as tested by direct
ELISA (4 nM; FIG. 1A). A competition ELISA was used to assess the
ability of RK35 to inhibit GDF-8 binding to its high affinity
receptor, ActRIIB RK35 blocked binding of biotinylated GDF-8 to
immobilized ActRIIB-Fc with an IC.sub.50.about.2.5 nM (FIG. 1B).
Soluble ActRIIb also blocked binding of GDF-8 to immobilized
ActRIIb-Fc while control antibodies did not block binding. The
neutralization activity of RK35 was also measured using a
pGL3-(CAGA)12 cell based reporter assay. In this assay the
luciferase gene was cloned under control of GDF-8/TGF-.beta.
responsive promoter and human A204 rhabdomyosarcoma cells were
transiently transfected with the reporter plasmid. Increases in
luciferase activity in A204 cells induced by GDF-8 were blocked in
a dose dependent manner by RK35 (FIG. 1C). RK35 reduced the GDF-8
signal transduction activity with an IC50 of 0.2 nM. Therefore,
RK35 is a new highly potent murine monoclonal neutralizing antibody
directed against GDF-8.
Example 3
In Vivo Activity of RK35 in Wild Type and ALS Rodent Models
Example 3.1
Experimental Procedures
Example 3.1.1
Animals and Drug Treatment
All procedures involving animals were approved by the IACUC of
either the University of Pennsylvania or Wyeth. Transgenic mice
expressing human SODG93A (Gurney et al., supra) on a B6SJL hybrid
background (Jackson Laboratories) were mated in-house to B6SJLF1
female breeder mice (Jackson Laboratories). Progeny were screened
by PCR; mice negative for the transgene were used as aged-matched
littermate wild type controls. Mice were divided into four groups:
29 SODG93A mice treated with the anti-myostatin antibody RK35, 28
SODG93A mice treated with phosphate-buffered saline (PBS)
(vehicle), 23 wild type mice treated with RK35, and 23 wild type
mice treated with PBS. Starting at 28 days after birth, mice were
intraperitoneally injected on a weekly basis, with either
anti-GDF-8 monoclonal antibody RK35 or an equivalent volume of PBS.
The first dose was 40 mg/kg; subsequent doses were 20 mg/kg/week
following the protocol described by for the anti-myo statin
antibody JA16 (Whittemore, et al., supra). 9-12 mice from each
group were sacrificed between 84 and 90 days (12 weeks) in age
(mean of 88 days) to assess wet muscle mass and histology, and the
remaining mice were monitored until reaching end-stage disease
(-134 days), defined as a failure to right within 30 sec from both
left and right lateral recumbency.
In parallel, transgenic rats (58) expressing human SODG93A (Howland
et al., supra) in an equal mix of males and females were
administered either PBS (vehicle) or RK35. Ten rats in each group
were euthanized at 95 days in age to determine the effect of RK35
on wet muscle mass. The remaining 19 rats in each group continued
through to end-stage disease. A second study using female
transgenic and wild type littermate control rats was used to
compare body weight increases as well as grip strength changes
across treatment groups and genotype. In each study, rats were
intraperitoneally injected with RK35 at 40 mg/kg (at 6 weeks (-42
days) of age) and subsequently injected with 20 mg/kg/week or
vehicle continuing until either sacrifice at 95 days to analyze
muscle mass, or end-stage as measured by right reflex failure.
Example 3.1.2
Body Weight and Muscle Mass Measurements
Initial body weights were used to evenly distribute animals among
cohorts so as to ensure equivalent average body weights at the
start of the study. Onset of weight loss was scored as the age at
which the first of three consecutive measures of weight loss was
observed. Wet muscle mass was determined on the gastrocnemius, the
cranial tibialis, the quadriceps, and the diaphragm at a point
consistent with early disease (88 days for mice, 95 days for rats)
and at end-stage (-134 days for mice and -128 days for rats).
Animals were euthanized and muscles from each leg were
quantitatively dissected and weighed; values from right and left
legs were averaged.
Example 3.1.3
Muscle Histopathology and Motor Neuron Counts
Gastrocnemius and diaphragm were fixed and sectioned for H&E
staining (Howland et al. (2002) Proc. Natl. Acad. Sci. U.S.A.
99:1604-29). Scoring for atrophy and hypertrophy was performed by
two independent pathologists, blinded to sample identity. Fiber
diameters were measured by morphometry (Axiovision 4.3, Zeiss,
Thornwood, N.Y.) on gastrocnemius muscle (PBS- and RK35-treated
SODG93A mice and PBS-treated wild type mice) at 88 days and
end-stage, as well as for diaphragm muscle (PBS- and RK35-treated
SODG93A and PBS-treated wild type mice) and end-stage. Three
muscles per group were snap frozen in dry-ice-cooled isopentane,
cryosectioned at a thickness of 8 .mu.m, and immunostained using an
anti-laminin antibody (Sigma, St. Louis, Mo.; catalog number
L9393). Linear measurements of the maximum diameter of the minor
axis of at least two hundred fibers were taken, using Zeiss
Axiovision software. Fiber diameters were binned in 20 .mu.m
intervals, and frequency histograms were generated for each muscle
group.
Motor neuron counts were performed on 3 mice from each group (wild
type, PBS-treated SODG93A, and RK35-treated SODG93A) at both
early-stage and end-stage disease (total of 18 mice analyzed).
Spinal columns removed from decapitated mice were post-fixed in 4%
paraformaldehyde, then cords were dissected and post-fixed for an
additional 24 hours in 4% paraformaldehyde. Using MULTIBRAINT.TM.
technology (Neuroscience Associates, Knoxyille, Tenn.), 18 lumbar
spinal cords were embedded together on a single block, and
cross-sectioned at 50 .mu.m in the coronal plane along the entire
segment (.about.6 mm) of the lumbar enlargement. Every sixth
section (300 .mu.m) was stained with thionine NISSL to reveal cell
bodies (Bjugn (1993) Brain Res. 627:25-33; Kieran et al. (2004)
Nat. Med. 10:402-05). Counts were performed using two independent
approaches. First, ten spinal cord sections encompassing L3-5 for
each of 18 mice were analyzed by an observer blinded to sample
identity using a Zeiss Axioplan2 at 20.times. and 40.times.. Both
ventral horns from each section were counted, identifying large
healthy motor neurons by the presence of visible nucleoli as
described previously (Kieran et al., supra). The resulting data
were represented as the average number of large motor neurons per
ventral horn. Second, sections from the L3-L5 region were analyzed
stereologically using a Zeiss AXIOSKOP.RTM.2 equipped with a
motorized specimen stage, electronic microcator, and stereology
software (STEREO INVESTIGATOR.RTM. (MBF Bioscience, Williston,
Vt.), as described (West et al. (1991) Anat. Rec. 231:482-97;
Schmitz and H of (2000) J. Chem. Neuroanat. 20:93-114; Schmitz and
H of (2005) Neuroscience 130:813-31); .alpha.-motor neurons were
scored as neurons with a maximum projection area greater than 300
.mu.m.sup.2.
Example 3.1.4
Phenotypic Analysis
Grip strength measurements (Columbus Instruments, Columbus, Ohio)
were performed biweekly starting 28 days after birth on both the
front and hind limbs of treated and control mice (n=8-24) as
described (LaMonte et al. (2002) Neuron 34:715-24). Transgenic and
wild type rats were tested for forelimb grip strength twice weekly
using a Dunnett rat grip strength meter (MJS Technology, Stevenage,
Hertfordshire, England) in early disease phase (between 95 and 110
days after birth; n=10). Rats were also analyzed by rotorod (Ugo
Basile, Comerio, Italy) as well as monitored for abnormalities in
gait and degrees of limb mobility (data not shown).
Example 3.1.5
Electrophysiology
Electromyography (EMG) recordings and data analysis were performed
blinded to genotype and treatment group. Nembutal-anesthetized rats
maintained at 35-37.degree. C. body temperature were subjected to
needle EMG by inserting a concentric monopolar needle electrode
(9013R0011, Medtronic, Minn., USA) into the surgically-exposed
diaphragm muscle until bursts of EMG interference pattern appeared
with each inspiration. Electrical signals were acquired at 20 KHz
with a BIOPAC setup consisting of MP150 Data Acquisition Unit,
UIM100C Universal Interface Module, EMG100C Electromyography
module, and the Acknowledge software (BIOPAC Systems Inc, Goleta,
Calif.). The signals were first analyzed to remove 60 Hz artifact
and band-pass filtered between 500 and 1000 Hz to remove movement
artifacts due to breathing and to emphasize the motor-unit
discharges. EMG bursts were identified by rectifying the signal,
low-pass filtering at 10 Hz, and then detecting the times at which
the resulting envelope was greater than its mean value. Bursts that
lasted less than 100 ms were not counted, and nonburst periods that
lasted less than 100 ms were counted as parts of the surrounding
bursts. Finally, the spikes in the nonrectified signal were
detected using a peak detection threshold set equal to three times
the standard deviation of the signal amplitude during the nonburst
periods. Spike burst analysis was performed with custom software
written by K. C. McGill (Stanford University, CA), and the burst
spike rate (Hz) for each animal computed as the mean number of
spikes per burst divided by the mean burst duration.
Example 3.1.6
Data Analysis and Statistics
A two-factor-repeated measures ANOVA model was applied on body
weight data as well as all grip strength data using an SAS mixed
procedure. A two-factor ANOVA linear model was applied on muscle
mass data using the SAS GLM procedure. Electrophysiology data was
analyzed by the two sample t-test. For motor neuron count data, a
generalized linear model (GLM) assuming Poisson distribution was
used. Muscle fiber data were analyzed by ANOVA followed by Tukey's
multiple comparison test. Comparisons were considered statistically
significant when p values were less than 0.05; comparisons with
0.05<p<0.15 were noted as trends.
Example 3.2
Results
Example 3.2.1
RK35 Treatment Increased Body Weights of SODG93A Rodents but did
not Extend Survival
SODG93A mice were treated with the anti-myostatin antibody RK35
starting 28 days after birth and continuing to end-stage disease
(approximately 134 days after birth). RK35 treatment resulted in
significantly increased body weight from 40 to 120 days after birth
compared to PBS-treated SODG93A mice. While PBS-treated SODG93A
mice reached a maximum body weight of 27.78.+-.0.46 g at 70 days,
RK35-treated mice reached a maximum of 32.13.+-.0.48 g, a relative
increase of 16% (FIG. 2A). While wild type mice continued to gain
weight throughout the study, both PBS- and RK35-treated SODG93A
mice began to show significant signs of weight loss due to disease
progression approximately 98 days after birth.
Transgenic SODG93A rats were also treated with RK35 antibody
starting 42 days after birth and continuing to end-stage disease
(.about.128 days old). Treatment with anti-myostatin RK35 led to
increased body weight in SODG93A rats compared to PBS-treated rats
(FIG. 2B). Transgenic rats receiving RK35 weighed significantly
more than PBS-treated transgenic rats as early as 60 days after
birth (p<0.05), corresponding to 3 weeks after initiation of
dosing. RK35-treated male rats reached a maximum of 458.8.+-.6.5 g
at 96 days, a 10% increase over PBS-treated male rats at
419.1.+-.10.7 g. RK35-treated females reached a maximum of
289.7.+-.9.3 g, a 15% increase over PBS-treated females at
252.8.+-.6.0 g. SODG93A rats treated with either PBS or RK35 began
to show significant weight loss after .about.112 days due to
disease progression; RK35 treatment did not delay the initiation of
weight loss.
While RK35 treatment led to significant increases in body weight,
the inventors observed no effects of RK35 treatment on survival.
Time to end-stage, as measured using the defined endpoint of
failure to right within 30 seconds, was 132.+-.8 days for
RK35-treated SODG93A mice (n=16), while PBS-treated SODG93A mice
reached end-stage by 134.+-.7 days (n=17). SODG93A rats treated
with RK35 reached end-stage by 125.+-.8 days compared to 128.+-.6
days for PBS-treated SODG93A rats. None of these differences were
statistically significant, indicating that inhibition of myostatin
does not delay time to end-stage disease in either mouse or rat
models of ALS.
Example 3.2.2
Effects of Myostatin Inhibition on Muscle Mass and Strength
In order to determine whether myostatin inhibition slowed muscle
wasting, SODG93A transgenic and wild type mice from each group were
sacrificed at 88 days after birth, a time point close to the
maximum increase in body weight induced by the RK35 treatment. At
this time point, PBS-treated SODG93A mice show significant
decreases in muscle mass in the gastrocnemius, cranial tibialis,
and quadriceps relative to wild type control mice consistent with
early-stage disease (FIG. 2C). In contrast, RK35-treated SODG93A
mice displayed statistically significant improvements in muscle
mass in all muscles examined in comparison to age-matched
PBS-treated SODG93A mice at the 88-day time point ranging from
.about.19% to 32% (gastrocnemius muscle, +26%; cranial tibialis,
+19%; quadriceps, +32%). While no significant loss of muscle mass
was observed in diaphragms from PBS-treated SODG93A mice during
early-stage disease; RK35 treatment induced a significant increase
in mass in this muscle as well (+21%).
The remaining mice in each cohort were monitored until end-stage as
defined by the right reflex test. At end-stage, leg muscle wasting
was observed in both RK35-treated and PBS-treated SODG93A mice
(FIG. 2E). In contrast to the observations on tissue from 88-day
mice, at end-stage disease PBS-treated SODG93A mice showed a
significant reduction in diaphragm muscle mass relative to wild
type animals (FIG. 2E). However, the RK35-induced increase in
diaphragm mass observed in treated SODG93A mice remained
significant at end-stage, indicating that myostatin inhibition
slowed atrophy of the diaphragm in the SODG93A mouse.
The effects of anti-myostatin antibody on muscle mass were also
investigated in SODG93A rats. Similar to observations made in mice,
.about.95-day-old SODG93A rats treated with RK35 showed
significantly increased mass over PBS-treated SODG93A rats in
gastrocnemius (+17%), cranial tibialis (+30%), quadriceps (+30%)
and diaphragm (+17%) muscles (FIG. 2D). By end-stage disease, leg
muscle atrophy was apparent in both PBS- and RK35-treated SODG93A
rats (FIG. 2F). A trend toward increased leg muscle mass as a
result of RK35 treatment in the SODG93A rats persisted to
end-stage, but these effects did not reach significance. However, a
robust and significant 35% increase in diaphragm mass from
RK35-treated SODG93A rats over PBS-treated controls (.about.35%)
was still evident at end-stage disease (FIG. 2F), in agreement with
the observations seen with the SODG93A mice (FIG. 2E).
In order to test the effects of myostatin inhibition on muscle
function, quantitative grip strength assays on RK35 and PBS-treated
mice were performed. Both RK35-treated and PBS-treated SODG93A mice
showed developmental increases in hind limb grip strength between
28 and 56 days after birth. By 56 days after birth, PBS-treated
SODG93A mice become significantly weaker than age-matched control
mice (FIG. 3A) (p<0.0001). While declines in grip strength were
also apparent in RK35-treated SODG93A mice by 63 days after birth,
the RK35-treated mice remained significantly stronger (or tended
toward stronger) than PBS-treated SODG93A mice from 49 to 88 days
after birth (p<0.05). Analysis of forelimb grip strength showed
a similar pattern (FIG. 3B). Peak forelimb strength in both PBS-
and RK35-treated SODG93A mice was observed by .about.49 and 56 days
after birth, respectively. PBS-treated SODG93A mice became
significantly weaker than wild type animals by 56 days after birth
(p<0.05) and continued to decline thereafter. RK35-treated
SODG93A mice were stronger than PBS-treated SODG93A mice from 56 to
88 days in age (56d; p=0.08; 63d; p=0.06; 70-88d; p<0.001).
These data indicate that myostatin inhibition slows loss of muscle
function through early-stage disease in the SODG93A mice. However,
after .about.100 days, declines in grip strength were similar in
both treated and untreated SODG93A mice.
To determine if RK35 treatment induced similar changes in SODG93A
rats, forelimb grip strength was also analyzed in RK35 and
PBS-treated SODG93A rats and age matched wild type littermates
during early-stage disease (FIG. 3C). PBS-treated SODG93A rats were
significantly weaker than wild type controls, confirming that
SODG93A rats also exhibit an early disease phase that precedes
overt motor deficits and weight loss in a manner similar to mice.
Grip strength measurements of RK35-treated SODG93A rats were
generally lower than those of PBS-treated wild type rats, although
the groups were not significantly different. Myostatin inhibition
in SODG93A rats did significantly improve grip strength when
compared to PBS-treated SODG93A rats at this age interval.
Example 3.2.3
Myostatin Inhibition Slowed the Degeneration of Limb Muscles and
Diaphragm in SODG93A Mice and Rats
The effects of RK35 treatment on muscle morphology were also
examined. Diaphragm and medial gastrocnemius muscle from 88 day
(early-stage disease) and .about.134 day (end-stage disease)
SODG93A mice were examined, comparing the effects of RK35 treatment
with PBS, in parallel with tissue from age-matched wild type
controls. The degree of atrophy and hypertrophy in each tissue was
scored in a blinded analysis (0, none; 1, slight; 2, mild; 3,
moderate; 4, marked; 5, severe) as shown in Table 4. PBS-treated
SODG93A mice showed significant atrophy of gastrocnemius at
early-stage disease (mean score of 2.0; and FIG. 4B). The observed
shrinkage of muscle fibers, centrally placed nuclei and
chromatin-condensed nuclei were consistent with muscle undergoing
active denervation; there was also evidence of inflammation
compared to wild type mice (FIGS. 4A and B). In contrast, the
gastrocnemius from early-stage SODG93A mice treated with RK35 (FIG.
4C) showed little to no atrophy (Table 4; mean score of 0.3). These
results support the muscle mass data indicating that atrophy of the
skeletal leg muscle in early phase disease (88 days after birth) in
the SODG93A mice is significantly reduced by myostatin
inhibition.
Examination of gastrocnemius from end-stage SODG93A mice confirmed
moderate muscle atrophy in both PBS (Table 4; mean score of 3.0)
and RK35 treated (Table 4; mean score of 3.3) (FIGS. 4E and F)
groups with no degenerative signs in wild type muscle (FIG. 4D).
These results are consistent with the muscle mass data, indicating
that the protective effects of myostatin inhibition in early
disease do not persist through end-stage disease in SODG93A
mice.
The diaphragm from either PBS-treated or RK35-treated SODG93A mice
showed little to no atrophy (Table 4; mean scores of 0.6) compared
to wild type mice at 88 days. By end-stage, however, mild to
moderate atrophy was observed in diaphragm from PBS-treated SODG93A
mice (FIG. 4H; Table 4; mean score of 2.3). In contrast, diaphragm
from RK35-treated SODG93A mice analyzed at end-stage disease showed
no significant signs of atrophy compared to PBS-treated SODG93A
animals (Table 4; compare FIGS. 4H and 4I), similar to diaphragm
from age-matched wild type mice (FIG. 4G). Taken together, the
muscle mass and histological assessment indicate that myostatin
inhibition by RK35 preserves diaphragm but not skeletal leg muscle
integrity through end-stage of disease.
TABLE-US-00004 TABLE 4 Summary of muscle pathology observed in
SODG93A mice treated with PBS or RK35, in comparison with
age-matched wild type control mice. genotype: G93A G93A WT G93A
G93A WT RK35: - + - - + - Age: 88 d 88 d 88 d 134 d 134 d 134 d #
mice: 3 3 3 3 3 3 gastrocnemius hypertrophy 0 0 0 0 0 0 atrophy 2
0.3 0 3.0 3.3 0 diaphragm hypertrophy 0 0.6 0 0 1.7 0 atrophy 0.6
0.6 0 2.3 0 0 Scoring: 0, none; 1, slight; 2, mild; 3, moderate; 4,
marked; 5, severe
Analysis of muscle fiber size in gastrocnemius and diaphragm muscle
showed a similar pattern. Frequency distributions of fiber diameter
measurements from 88-day gastrocnemius muscle show a shift toward
smaller fibers in SODG93A mice (FIG. 5A) in comparison to wild type
control mice (FIG. 5C). The distribution of fiber diameters from
RK35-treated SODG93A mice (FIG. 5B) is intermediate between
untreated SODG93A mice and wild type mice during early-stage
disease. By end-stage, however, average fiber diameter in the
gastrocnemius muscle of RK35-treated SODG93A mice did not differ
significantly from PBS-treated SODG93A mice (data not shown).
Average fiber diameters in the gastrocnemius muscle from both
RK35-treated and PBS-treated SODG93A mice were significantly
different than wild type at end-stage, consistent with the marked
muscle atrophy observed by histology. Significant differences in
fiber size between wild type and PBS-treated SODG93A mice were also
evident at end-stage in diaphragm muscle. Diaphragm muscle fiber
size from RK35-treated SODG93A mice showed a significant shift in
average fiber diameter, leading to a size distribution intermediate
between wild type and PBS-treated SODG93A mice (FIG. 5D).
The effects of SODG93A expression on the electrical activity of the
diaphragm muscle (FIGS. 4J and 4K) were next examined using the rat
model at a time corresponding to clinical onset (.about.112 days;
Howland et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:1604-09).
The EMG shown for the PBS-treated SODG93A rat group shows sparse
spike activity as well as some presence of abnormal spontaneous
activity (FIG. 4J). Spike activity of SODG93A rats treated with
RK35 was similar to that observed for wild type rats, with no
evidence of abnormal spontaneous activity. As shown in FIG. 4K,
diaphragm muscle from transgenic SODG93A rats showed a
statistically significant decrease in EMG burst spike rates
indicative of impaired function. In contrast, SODG93A rats treated
with RK35 showed a burst spike rate that was significantly higher
than the PBS-treated SODG93A rats, and which was not significantly
different from age-matched wild type controls. Therefore, myostatin
inhibition by RK35 was effective in preserving both diaphragm
structure and diaphragm function.
Example 3.2.4
Myostatin Inhibition Slows Loss of Motor Neurons in the Ventral
Horn
To determine whether RK35 slowed the loss of large motor neurons in
spinal cord, large motor neurons in lumbar L3-5 mouse spinal cord
from SODG93A mice treated with RK35 or PBS and wild type mice
treated with PBS were counted. Counts were performed on spinal cord
at 12 weeks (84-90 days in age; mean of 88 days), a time when RK35
treatment resulted in increased muscle mass, increased body weight,
increased grip strength and attenuated muscle histopathology in
SODG93A mice. At this time, there is significant loss (25-40%) of
large motor neurons in the SODG93A mouse (Guo et al. (2003) Hum.
Mol. Genet. 12:2519-32; Sharp (2005) Neuroscience 130:897-910;
Schutz et al. (2005) J. Neurosci. 25:7805-12).
Counts of large lumbar motor neurons (FIGS. 6A-D) in PBS-treated
SODG93A mice (FIG. 6F) decreased significantly compared to
littermate wild type controls (FIG. 6E). RK35 treatment reduced the
loss of motor neurons at early-stage disease (FIG. 6G) to a level
intermediate between PBS-treated SODG93A mice and wild type
mice.
By end-stage disease, significant losses of large motor neurons in
the lumbar ventral horn as well as increased gliosis were evident
in the SODG93A mice regardless of treatment (FIGS. 6I and J) with
no corresponding changes noted in wild type mice (FIG. 6H).
Stereological counting of the total population of large motor
neurons (area greater than 300 .mu.m.sup.2) revealed a trend of 25%
motor neuron loss in PBS-treated SODG93A mice in comparison to wild
type controls. A trend toward a slowing of motor neuron loss was
observed in mice treated with RK35 (p=0.08) (FIG. 6A). If counts
were restricted to large motor neurons with visible nucleoli to
avoid counting motor neurons showing signs of degeneration (i.e.,
presence of irregular membrane and vacuoles), there is a 40%
decrease in the average number of large motor neurons per section
in PBS-treated SODG93A mice in comparison to wild type control mice
(FIG. 6B), as well as a statistically significant difference
between RK35-treated and PBS-treated SODG93A mice (FIG. 6B). Taken
together, however, the composite data shown in FIGS. 6A and B
indicate that both the loss of large motor neurons and the effects
of RK35 treatment on this loss are relatively subtle at the 12-week
(88 day) age interval.
By end-stage, motor neuron counts in both RK35-treated and
PBS-treated SODG93A mice were significantly different from wild
type control mice by both methods of analysis (FIGS. 6C and D).
These data are consistent with data on skeletal muscle structure
and function presented above that the RK35-mediated improvements
observed during early-stage disease are not maintained at
end-stage.
Example 4
Discussion
ALS is a fatal and progressive disease in which motor neurons of
the spinal cord and brain stem degenerate with subsequent muscle
atrophy. Considerable attention has focused on mechanisms involved
in motor neuron cell death. Several recent studies have suggested
that multiple cell types may be involved in the etiology of the
disease by controlling the production of key factors in the
extracellular microenvironment of the neuromuscular junction
(Bruijn et al. (2004) Annu. Rev. Neurosci. 27:723-49). Studies
using chimeric mice have shown that the presence of wild type
normeuronal cells can extend survival of motor neurons expressing
mutant SOD1 (Clement et al. (2003) Science 302:113-17). These
observations have led to the investigation of therapies that might
slow neuronal degeneration by providing an optimal microenvironment
for survival. For example, administration into muscle of virally
expressed growth factors including IGF-1, GDNF and VEGF have all
been shown to prolong survival in the SODG93A mouse model (Kaspar
et al. (2003) Science 301:839-42; Azzouz et al. (2004) Nature
429:413-17; Wang et al (2002) J. Neurosci. 22:6920-28).
Furthermore, muscle-specific expression of IGF-1 has been shown to
stabilize neuromuscular junctions, enhance motor neuron survival,
and delay onset and progression of disease in the SODG93A
transgenic mouse model, indicating that direct effects on muscle
can impact disease onset and progression (Dobrowolny et al. (2005)
J. Cell. Biol. 168:193-99). Changes in muscle metabolism and motor
neuron vulnerability have also been reported in ALS mice, further
supporting the hypothesis that muscle may be an active driver of
disease pathology (Dupois et al. (2004) Proc. Natl. Acad. Sci. USA
101:11159-64).
Myostatin, or GDF-8, is an endogenous inhibitor of muscle growth,
eliciting its biological function, at least in part, by activation
of the Activin lib receptor (ActRIIb), resulting in repression of
myoblast cell proliferation and differentiation (Langley et al.
(2002) J. Biol. Chem. 277:49831-40; Thomas et al. (2000) J. Biol.
Chem. 275:40235-43). Inhibition of GDF-8 function using anti-GDF-8
neutralizing antibodies has been shown to enhance muscle mass and
strength in healthy adult mice as well as provide functional
improvement in the mdx mouse model of muscular dystrophy
(Whittemore et al. (2003) Biochem. Biophys. Res. Commun.
300:965-71; Bogdanovich et al. (2002) Nature 420:418-21). To better
understand the role of muscle in motor neuron disease progression,
a novel neutralizing antibody to GDF-8, RK35, which binds with
higher affinity than a previously described reagent (IC.sub.50 3 nM
for RK35 vs. >100 nM for JA16; Whittemore et al. (2003) Biochem.
Biophys. Res. Commun. 300:965-71; Bogdanovich et al. (2002) Nature
420:418-21) was used, resulting in greater increases in muscle mass
in wild type mice treated with RK35 (data not shown).
In SODG93A mouse and rat models of familial ALS, treatment with
RK35 resulted in increased body weight and increased muscle mass
and strength during the early phases of motor neuron disease. This
early phase of disease is defined as the age (56-88 days after
birth) at which SODG93A mice show a significant loss of muscle
strength, as measured by grip strength assessment (Ligon et al.
(2005) Neuroreport 16:533-36) and gait abnormalities (Wooley et al.
(2005) Muscle Nerve 32:43-50), and which coincides with the
denervation of neuromuscular junctions (Frey et al. (2000) J.
Neurosci. 20:2534-42; Fischer et al (2004) Experimental Neurology
185:232-40). Muscle mass increases resulting from GDF-8 inhibition
by RK35 were most evident in the quadriceps muscles, but were also
pronounced in the gastrocnemius, cranial tibialis and the diaphragm
in both rodent models tested. These increases correlated well with
increased strength, as hindlimb and forelimb strength declined more
slowly in RK35-treated mice in comparison to controls. The extent
of muscle mass increase induced by treatment with the RK35
anti-GDF-8 antibody was similar in magnitude to a 25% increase in
muscle mass observed in mice heterozygous for disruption of the
GDF-8 gene; muscle mass from mice that are homozygous null for
GDF-8 is about two-fold that of wild type mice (McPherron (1997)
Nature 387:83-90).
At approximately 84-88 days after birth in SODG93A mice, and
approximately 110 days for SODG93A rats, overt signs of disease
including body weight decreases, gait abnormalities and paralysis
become evident. RK35 treatment did not extend survival in either
SODG93A mice or rats. The increased muscle mass and strength
induced by anti-GDF-8 treatment in the early phase of disease did
not delay the appearance of gait abnormalities and limb paralysis
in both SODG93A mice and rats (data not shown), nor were gains in
leg muscle mass maintained.
However significant increases in diaphragm mass in RK35-treated
SODG93A mice and rats as compared to vehicle-treated SODG93A
controls in both early and end-stage disease phases were observed.
Diaphragm muscle in RK35-treated SODG93A mice at end-stage was
comparable to that of age-matched wild type controls in both mass
and histological evaluation. RK35-treated SODG93A rats also
maintained a significant increase in diaphragm muscle mass at
end-stage. Consistent with these morphological changes,
electrophysiological analysis of diaphragms from untreated SODG93A
rats indicates that expression of mutant SOD1 results in
significant inhibition of muscle function, and that treatment with
RK35 effectively preserved muscle function in the diaphragm.
In this study a defined endpoint (failure of the right reflex test,
indicating significant limb paralysis) was used, as the criteria
for "end-stage" and euthanasia. Therefore it is not clear whether
the enhanced diaphragm muscle mass, decreased atrophy, and improved
electrophysiological function induced by RK35 treatment would have
resulted in prolonged lifespan in rodents provided with nutritional
supplementation. However, these findings are potentially important
given the fact that respiratory dysfunction is the leading cause of
death in patients with ALS (Lechtzin et al. (2002) Amyotroph.
Lateral Scler. Other Motor Neuron Disord. 3:5-13). Treatments such
as RK35 designed to enhance diaphragm function may have the
potential to delay the necessity for mechanically assisted
breathing in ALS patients.
GDF-8 inhibition by RK35 may influence motor neuron loss in the
lumbar spinal cord in the early disease phase, although many
therapeutic benefits apparently were lost by end-stage disease.
These data indicate that therapies acting directly on muscle can
have a benefit on motor neurons innervating muscle, possibly by
modulating the trophic microenvironment, although this approach is
apparently not sufficient to delay disease. These observations are
therefore consistent with the results of Dobrowolny et al. ((2005)
J. Cell. Biol. 168:193-99), in which expression of a
muscle-specific isoform of IGF led to slowed loss of motor neurons
in the SODG93A mouse model, and that of Kaspar et al. ((2003)
Science 301:839-42), where viral delivery of IGF1 to muscle
resulted in reduced motor neuron loss in early-stage disease.
Similar to the observations of Kaspar et al. ((2003), supra),
beneficial effects of treatment on motor neuron survival were not
maintained through end-stage disease. Improved trophic factor
support from muscle is therefore likely to be insufficient to
prevent motor neuron loss in the SODG93A model.
In summary, in both mouse and rat models of familial ALS,
inhibition of myostatin results in enhanced muscle mass and
strength, which is maintained through the early stages of disease
but lost by end-stage. Myostatin inhibition slowed degenerative
changes in skeletal muscle in early-stage disease, but did not
delay onset of paralysis nor extend survival, as defined by right
reflex failure, nor did myostatin inhibition significantly slow
motor neuron loss. However, both morphological and functional
differences through late-stage disease were observed in the
diaphragm muscle of animals treated with anti-myostatin antibody,
in comparison to untreated controls. Overall, the data provided
herein support the potential for a beneficial effect of muscle
building by treatment with RK35, which may contribute to an
enhanced "quality of patient life" early in the disease process.
Given that anti-GDF-8 antibodies are currently in clinical
development, use of such clinical reagents in ALS for the
maintenance of limb and diaphragm muscle mass warrants further
investigation as a component of a multi-pronged approach to the
treatment of ALS. The combination of an anti-GDF-8 therapy with
existing drugs such as the glutamate-antagonist riluzole, or newer
agents entering clinical development, might not only improve the
level of efficacy by helping maintain muscle mass but also have
significant impact on overall patient quality of life.
Example 5
Mapping of Epitopes for RK35
In order to map the exact antibody epitopes to GDF-8, 48
overlapping 13-residue peptides representing the entire sequence of
mature GDF-8 set forth in SEQ ID NO:1 were synthesized directly on
cellulose paper using the spot synthesis technique (e.g., Molina et
al. (1996) Peptide Res. 9:151-55; Frank et al. (1992) Tetrahedron
48:9217-32). The overlap of the peptides was 11 amino acids. In
this array, cysteine residues were replaced with serine in order to
reduce the chemical complications caused by cysteines. Cellulose
membranes modified with polyethylene glycol and Fmoc-protected
amino acids were purchased from Abimed (Lagenfeld, Germany). The
array was defined on the membrane by coupling a .beta.-alanine
spacer, and peptides were synthesized using standard DIC
(diisopropylcarbodiimide)/HOBt (hydroxybenzotriazole) coupling
chemistry as described previously (Molina et al., supra; Frank et
al., supra).
Activated amino acids were spotted using an Abimed ASP 222 robot.
Washing and deprotection steps were done manually, and the peptides
were N-terminally acetylated after the final synthesis cycle.
Following peptide synthesis, the membrane was washed in methanol
for 10 minutes and in blocker (TBST (Tris-buffered saline with 0.1%
(v/v) TWEEN.TM. 20) and 1% (w/v) casein) for 10 minutes. The
membrane was then incubated with 2.5 .mu.g/ml of an anti-GDF-8
antibody in blocker for 1 hr with gentle shaking After washing in
blocker 3 times for 10 min, the membrane was incubated with
HRP-labeled secondary antibody (0.25 .mu.g/ml in blocker) for 30
min. The membrane was then washed three times for 10 min each with
blocker and 2 times for 10 minutes each with TBST. Bound antibody
was visualized using SUPERSIGNAL.TM. West reagent (Pierce;
Rockford, Ill.) and a digital camera (Alpha Innotech Fluorimager
(Alpha Innotech; San Leandro, Calif.)). As shown in FIG. 7, the
RK35 epitope maps to a region of GDF-8 between amino acids 30-40
and 84-97, that putatively interacts with the GDF-8 Type II
receptor as predicted by GDF-8 receptor sequence comparison with
homologous TGF-.beta. family receptors with characterized
domains.
Example 6
Characterizing the RK35 Antibody
The variable heavy (VH) and variable light (VL) genes encoding RK35
were cloned from hybridoma cells producing the antibody, and the
amino acid sequences were determined. Sequence data for the RK35
antibody was used to identify the nearest germline sequence for the
heavy and light chain. A comparison of RK35 light and heavy
variable regions with the closest human germline sequences is shown
in FIG. 8. Appropriate mutations may be made using standard
site-directed mutagenesis techniques with the appropriate mutagenic
primers. Mutation of the antibody is then confirmed by sequence
analysis. Exemplary amino acid sequences for humanized RK35 are set
forth in SEQ ID NOs:7 (VH) and 9 (VL), both of which may be encoded
by a nucleic acid sequence readily determined by a skilled artisan,
e.g., the nucleic acid sequences set forth as SEQ ID NO:6 (VH) and
8 (VL). A skilled artisan will recognize that any and/or all amino
acids in the framework of the humanized antibody may be mutated
back to the original murine amino acid, e.g., to maintain the
conformation of the antigen binding fragment. Nonlimiting examples
of SEQ ID NO:7 (VH) and SEQ ID NO:9 (VL) with some back-mutations
that may help to maintain the affinity of the antibody to GDF-8 are
set forth as SEQ ID NO:26 and SEQ ID NO:27, respectively.
To create chimeric antibodies, the VH sequence is subcloned into a
pED6 huIgG1 mut expression vector, which encodes human IgG1
containing two point mutations (L234A and G237A) to reduce binding
to human Fc receptors and complement components (Morgan et al.
(1995) Immunology 86:319-24; Shields et al (2001) J. Biol. Chem.
276:6591-604). The VL sequence of RK35 may be subcloned into the
pED6 Kappa expression vector. The expression vectors containing the
RK35 VH and VL sequences are then cotransfected into COS-1 cells
and a chimeric RK35 antibody is purified from conditioned
medium.
Example 7
Treatment of Muscle Disorders
Inhibitors of GDF-8, i.e., GDF-8 antagonists, such as, for example,
inhibitory antibodies, are useful for treatment of metabolic
disorders associated with GDF-8 such as type 2 diabetes, impaired
glucose tolerance, metabolic syndrome (e.g., syndrome X), insulin
resistance (e.g., induced by trauma such as burns or nitrogen
imbalance), and adipose tissue disorders (e.g., obesity) Inhibitors
of GDF-8 are also useful for the treatment of bone and muscle
disorders associated with GDF-8, such as ALS, muscular dystrophy
and osteoarthritis. The anti-GDF-8 antibodies and antibody
fragments of the invention may be used to treat a subject, e.g., a
human subject, preferably a subject suffering from ALS, at disease
onset, or a subject having an established metabolic or
bone/muscular disease. The inhibitory antibodies against GDF-8 may
also be used to prevent and/or to reduce the severity and/or the
symptoms of the disease. It is anticipated that the anti-GDF-8
antibodies and antibody fragments are administered, e.g.,
subcutaneously, as frequently as once per day and as infrequently
as once per month. Treatment durations range from about one month
(or less) to several years.
To test the clinical efficacy of anti-GDF-8 in humans, subjects
suffering from or at risk for ALS are identified and randomized to
treatment groups. Treatment groups include a placebo group and one
to three or more groups receiving antibody (at different doses when
there are two or more groups). Individuals are followed
prospectively for, e.g., one month to three years to assess changes
in weight, muscle mass, and grip strength. It is anticipated that
individuals receiving treatment will exhibit an improvement.
A GDF-8 antagonist, preferably in the form of an antibody or
antibodies, is administered as the sole active compound or in
combination with another compound or composition. When administered
as the sole active compound or in combination with another compound
or composition, the dosage is preferably from approximately 1
.mu.g/kg to 100 mg/kg, depending on the severity of the symptoms
and/or the progression of the disease. The appropriate effective
dose may be selected by a treating clinician from the following
nonlimiting list of ranges: 1 .mu.g/kg to 100 mg/kg, 1 .mu.g/kg to
50 mg/kg, 1 .mu.g/kg to 20 mg/kg, 1 .mu.g/kg to 10 mg/kg, 1
.mu.g/kg to 1 mg/kg, 10 .mu.g/kg to 1 mg/kg, 10 .mu.g/kg to 100
.mu.g/kg, 100 .mu.g to 1 mg/kg, and 500 mg/kg to 1 mg/kg. Exemplary
nonlimiting treatment regimens and potential outcomes are
summarized in Table 5.
TABLE-US-00005 TABLE 5 Examples of Potential Clinical Cases Status
prior to Treatment treatment Regimen Potential Outcome No clinical
signs, 0.01-1 mg/kg every 4 Prevention of ALS or family history of
weeks for 48 weeks delay of onset ALS Mild clinical signs 0.01-100
mg/kg weekly Improved grip of ALS for 4 or more weeks strength,
weight gain, and muscle mass Advanced stage of 0.01-100 mg/kg twice
Improvement of ALS characterized weekly for 6 or more clinical
signs, by severe muscle weeks reduction in severity wasting, weight
of symptoms and/or loss, and loss of increase in muscle strength,
etc. mass/body fat ratio
The specification is most thoroughly understood in light of the
teachings of the references cited within the specification, all of
which are hereby incorporated by reference herein in their
entireties. The embodiments within the specification provide
illustrations of the invention and should not be construed to limit
the scope of the invention. The skilled artisan recognizes that
many other embodiments are encompassed by the claimed invention,
and that the specification and examples should be considered as
exemplary only.
SEQUENCE LISTINGS
1
341109PRTHomo sapiens 1Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr
Glu Ser Arg Cys Cys 1 5 10 15 Arg Tyr Pro Leu Thr Val Asp Phe Glu
Ala Phe Gly Trp Asp Trp Ile 20 25 30 Ile Ala Pro Lys Arg Tyr Lys
Ala Asn Tyr Cys Ser Gly Glu Cys Glu 35 40 45 Phe Val Phe Leu Gln
Lys Tyr Pro His Thr His Leu Val His Gln Ala 50 55 60 Asn Pro Arg
Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser 65 70 75 80 Pro
Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly 85 90
95 Lys Ile Pro Ala Met Val Val Asp Arg Cys Gly Cys Ser 100 105
2348DNAMus musculus 2gaagtgcagc tggtggagtc tgggggaggc ttagtgaagc
ctggagggtc cctgaaactc 60tcctgtgcag cctctggatt cactttcagt agctatgcca
tgtcttgggt tcgccagact 120ccggagaaga ggctggagtg ggtcgcaacc
attagtagtg gtggtagtta cacctcctat 180ccagacagtg tgaagggtcg
attcaccatc tccagagaca atgccaagaa caccctgtac 240ctgcaaatga
gcagtctgag gtctgaggac acggccatgt attactgtgc aagacaagac
300tatgctatga actactgggg tcaaggaacc tcagtcaccg tctcctca
3483116PRTMus musculus 3Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln
Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser
Gly Gly Ser Tyr Thr Ser Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90
95 Ala Arg Gln Asp Tyr Ala Met Asn Tyr Trp Gly Gln Gly Thr Ser Val
100 105 110 Thr Val Ser Ser 115 4321DNAMus musculus 4gacattgaga
tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60atcacctgca
aggccagtca ggatgtgagt actgctgtag cctggtatca acagaaacca
120ggacaatctc ctaaactact gctttactcg gcatcctacc ggtacactgg
agtccctgat 180cggttcactg gcagtggatc tgggacggat ttcactttca
ccatcagcag tgtgcaggct 240gaagacctgg cagtttatta ctgtcagcaa
cattatagta ctccgtggac gttcggtgga 300ggcaccaagc tggaaatcaa a
3215107PRTMus musculus 5Asp Ile Glu Met Thr Gln Ser His Lys Phe Met
Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala
Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ser Pro Lys Leu Leu Leu 35 40 45 Tyr Ser Ala Ser Tyr
Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu
Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp 85 90
95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
6348DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 6gaggtccaac tattagaatc gggaggcggt
ctggttcagc caggagggag tctcaggctt 60agttgcgctg cgtcgggatt caccttttca
agttacgcaa tgtcatgggt tcgtcaggca 120ccggggaaag gcttagagtg
ggtgtcaact attagctctg gcggtagcta tacgtcgtat 180cctgactctg
tgaagggacg atttacaata agccgggaca attctaaaaa cactttgtac
240ctacagatga attccttgag agccgaagat accgccgtct actattgtgc
gaagcaagat 300tacgctatga actattgggg tcaagggaca atggtaacgg tatcctcc
3487116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 7Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Ser
Gly Gly Ser Tyr Thr Ser Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Lys Gln Asp Tyr Ala Met Asn Tyr Trp Gly Gln Gly Thr Met Val
100 105 110 Thr Val Ser Ser 115 8321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
8gacattcaaa tgacccaaag cccttcttcc ttaagcgcat cagtaggtga ccgagttaca
60ataacgtgta aagcgagcca agatgtgagt actgcagtag cgtggtatca gcaaaagcca
120gggaaggctc cgaaactatt gatttactcc gcctcttaca gatatacggg
cgttcctgat 180aggtttagtg gaagtgggtc gggtacggac tttaccctga
ctatatcgtc acttcagcca 240gaggattttg ctacctacta ttgccaacag
cattattcaa caccgtggac attcggccag 300ggaactaagg tcgaaataaa a
3219107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 9Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala
Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr
Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 105PRTMus
musculus 10Ser Tyr Ala Met Ser 1 5 1117PRTMus musculus 11Thr Ile
Ser Ser Gly Gly Ser Tyr Thr Ser Tyr Pro Asp Ser Val Lys 1 5 10 15
Gly 127PRTMus musculus 12Gln Asp Tyr Ala Met Asn Tyr 1 5 1311PRTMus
musculus 13Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala 1 5 10
147PRTMus musculus 14Ser Ala Ser Tyr Arg Tyr Thr 1 5 157PRTMus
musculus 15Gln Gln His Tyr Ser Thr Pro 1 5 16321DNAHomo sapiens
16cgaaccgtag cggcaccgtc agtattcata ttccctccat cggacgagca attgaaaagt
60gggacagctt cggtcgtgtg tctcttgaat aacttttacc ccagagaagc taaggtccag
120tggaaagttg acaatgcgtt acagagcgga aattctcaag aatccgttac
tgaacaggat 180agtaaggatt ctacgtattc acttagcagt actctgaccc
tatcaaaggc agattatgaa 240aaacacaagg tatacgcctg cgaggtgacg
catcaaggct tatccagccc agttacaaaa 300tcttttaaca ggggtgagtg t
32117107PRTHomo sapiens 17Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85
90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105
18990DNAHomo sapiens 18gcctccacaa aaggaccatc tgtttttccc ttggcgccat
caagtaaatc tacttccgga 60ggtaccgctg cgcttggctg cctcgtgaaa gactatttcc
cagaacctgt cacagtctcg 120tggaatagtg gtgctttaac ctcgggcgta
catacttttc ctgctgtact tcaatcaagc 180ggactgtact cattatcgtc
tgtagtcacg gtcccgagtt cttcactcgg aacacagact 240tatatatgca
acgttaatca taagcctagc aacaccaagg ttgacaaaaa ggtggaacca
300aaatcgtgcg ataagacgca cacatgtcca ccctgtcctg caccagaagc
tctgggcgcg 360ccatcggttt tcttgttccc acccaaacct aaggacacgt
taatgataag tcgaacgcca 420gaggtgacat gtgttgtagt ggatgtgagc
cacgaagatc cggaagtaaa attcaattgg 480tatgtagatg gtgttgaagt
ccataacgca aaaactaagc cgagggaaga gcagtacaac 540tctacttata
gggtagtctc cgtactaact gtcttgcacc aagattggct aaatgggaag
600gaatataaat gtaaggtttc aaataaggca ctaccagccc cgatagagaa
gacaataagc 660aaagcgaagg ggcaaccaag agagccccaa gtgtacacct
tgcctccgag cagagaggaa 720atgacaaaaa atcaagtatc ccttacgtgt
ctggtaaagg gattttatcc aagtgacata 780gcagtggagt gggagagtaa
cggccagcca gaaaacaatt acaaaaccac tcccccggtt 840ttagatagcg
atgggagttt ctttttgtac tcaaagttga ccgttgacaa gtcacgatgg
900cagcaaggta atgtatttag ttgttctgtt atgcatgagg ccttacataa
tcactacacg 960cagaaatctc tctccttaag ccccgggaaa 99019330PRTHomo
sapiens 19Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110
Pro Ala Pro Glu Ala Leu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro 115
120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235
240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 325 330 2010PRTMus musculus 20Gly Phe Thr Phe
Ser Ser Tyr Ala Met Ser 1 5 10 2110PRTMus musculus 21Thr Ile Ser
Ser Gly Gly Ser Tyr Thr Ser 1 5 10 227PRTMus musculus 22Gln Asp Tyr
Ala Met Asn Tyr 1 5 2311PRTMus musculus 23Lys Ala Ser Gln Asp Val
Ser Thr Ala Val Ala 1 5 10 247PRTMus musculus 24Ser Ala Ser Tyr Arg
Tyr Thr 1 5 257PRTMus musculus 25Gln Gln His Tyr Ser Thr Pro 1 5
26116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 26Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln
Ala Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser
Gly Gly Ser Tyr Thr Ser Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys 85 90
95 Ala Lys Gln Asp Tyr Ala Met Asn Tyr Trp Gly Gln Gly Thr Met Val
100 105 110 Thr Val Ser Ser 115 27107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
27Asp Ile Glu Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr
Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro
Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln His Tyr Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 28405DNAMus musculus 28atgaactttg
tgctcagctt gattttcctt gccctcattt taaaaggtgt ccagtgtgaa 60gtgcagctgg
tggagtctgg gggaggctta gtgaagcctg gagggtccct gaaactctcc
120tgtgcagcct ctggattcac tttcagtagc tatgccatgt cttgggttcg
ccagactccg 180gagaagaggc tggagtgggt cgcaaccatt agtagtggtg
gtagttacac ctcctatcca 240gacagtgtga agggtcgatt caccatctcc
agagacaatg ccaagaacac cctgtacctg 300caaatgagca gtctgaggtc
tgaggacacg gccatgtatt actgtgcaag acaagactat 360gctatgaact
actggggtca aggaacctca gtcaccgtct cctca 40529135PRTMus musculus
29Met Asn Phe Val Leu Ser Leu Ile Phe Leu Ala Leu Ile Leu Lys Gly 1
5 10 15 Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Lys 20 25 30 Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe 35 40 45 Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Thr
Pro Glu Lys Arg Leu 50 55 60 Glu Trp Val Ala Thr Ile Ser Ser Gly
Gly Ser Tyr Thr Ser Tyr Pro 65 70 75 80 Asp Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln
Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met 100 105 110 Tyr Tyr Cys
Ala Arg Gln Asp Tyr Ala Met Asn Tyr Trp Gly Gln Gly 115 120 125 Thr
Ser Val Thr Val Ser Ser 130 135 30381DNAMus musculus 30atggagtcac
agattcaggt ctttgtattc gtgtttctct ggttgtctgg tgttgacgga 60gacattgaga
tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc
120atcacctgca aggccagtca ggatgtgagt actgctgtag cctggtatca
acagaaacca 180ggacaatctc ctaaactact gctttactcg gcatcctacc
ggtacactgg agtccctgat 240cggttcactg gcagtggatc tgggacggat
ttcactttca ccatcagcag tgtgcaggct 300gaagacctgg cagtttatta
ctgtcagcaa cattatagta ctccgtggac gttcggtgga 360ggcaccaagc
tggaaatcaa a 38131127PRTMus musculus 31Met Glu Ser Gln Ile Gln Val
Phe Val Phe Val Phe Leu Trp Leu Ser 1 5 10 15 Gly Val Asp Gly Asp
Ile Glu Met Thr Gln Ser His Lys Phe Met Ser 20 25 30 Thr Ser Val
Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp 35 40 45 Val
Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro 50 55
60 Lys Leu Leu Leu Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp
65 70 75 80 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr
Ile Ser 85 90 95 Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys
Gln Gln His Tyr 100 105 110 Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys 115 120 125 3295PRTHomo sapiens 32Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ser Tyr Ser Thr Pro 85 90 95 3398PRTHomo sapiens 33Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys 3415PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 34Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10
15
* * * * *
References